- Email: [email protected]
Dantrolene is neuroprotective in vitro, but does not affect survival in SOD1G93A mice
Neuroscience 220 (2012) 26–31
DANTROLENE IS NEUROPROTECTIVE IN VITRO, BUT DOES NOT AFFECT SURVIVAL IN SOD1G93A MICE K. A. STAATS, a,b M. VAN RILLAER, a W. SCHEVENEELS, a,b R. VERBESSELT, c P. VAN DAMME, a,b,d W. ROBBERECHT a,b,d AND L. VAN DEN BOSCH a,b*
resulting in muscle weakness and paralysis. The disease has an annual incidence of 2.7 cases per 100,000 people in Europe (Logroscino et al., 2010) and most patients succumb to the disease within 3–5 years after onset. On average 10% of all ALS cases are familial, of which 20% are caused by mutations in superoxide dismutase 1 (SOD1). On the basis of mutations in SOD1, ALS rodent models have been generated that predictably mimic the patient disease process (Julien and Kriz, 2006). As disease progression is indistinguishable between familial and sporadic cases, common disease mechanisms are expected. One of these mechanisms is excitotoxicity, which entails the cell death of neurons by glutamatergic overstimulation. The only drug currently available for the disease, riluzole, targets this mechanism and prolongs survival of ALS patients (Miller et al., 2007). Overstimulation by glutamate opens calcium-permeable a-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and increases intracellular calcium concentrations which can become hazardous for motor neurons (Van Den Bosch et al., 2006). It is hypothesized that release of calcium from the endoplasmic reticulum (ER) may contribute to the vulnerability of motor neurons to excitotoxicity and that blocking this calcium release would be beneficial in ALS (Grosskreutz et al., 2010). A role for ER calcium in ALS has been indicated by the higher expression of the gene encoding the ER calcium channel inositol 1,4,5-trisphosphate receptor 2 (ITPR2) in total blood from ALS patients (van Es et al., 2007) and by mutations discovered in familial ALS in the charged multivesicular body protein 2b gene (CHMP2B), resulting in impaired ER calcium concentrations (Cox et al., 2010). An important calcium release channel present in the ER is the ryanodine receptor (RyR). This receptor allows calcium-induced calcium release from the ER and blocking the RyR receptor by dantrolene decreases calcium transients in cultured motor neurons (Jahn et al., 2006) and protects neurons in vitro (Rothstein and Kuncl, 1995; Wei and Perry, 1996; Hernandez-Fonseca and Massieu, 2005; Liu et al., 2009). In addition, dantrolene administration is beneficial in rodent models of a number of neurodegenerative conditions (Inan and Wei, 2010), including spinocerebellar ataxia type 2 (Liu et al., 2009) and type 3 (Chen et al., 2008), spinal cord injury (Aslan et al., 2009) and cerebral ischemia (Wei and Perry, 1996). By inhibiting the RyR with dantrolene, we investigate whether calcium entering the cell from the extracellular space can further increase the burden of calcium by the process of calcium-induced calcium release from the ER. We use primary motor neurons to confirm the protective
a
Laboratory of Neurobiology and Leuven research Institute for Neurodegenerative Disorders (LIND), KU Leuven, Belgium
b
Vesalius Research Center, VIB, Belgium
c
Center for Clinical Pharmacology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Belgium d
Department of Neurology, Gasthuisberg, Leuven, Belgium
Abstract—Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease. One of the proposed disease mechanisms is excitotoxicity, in which excessive cytosolic calcium causes neuronal death. Although most calcium may originate from the extracellular space through activation of calcium-permeable AMPA receptors, we investigated in this study the contribution of endoplasmic reticulum calcium release by blocking the ryanodine receptor (RyR) using dantrolene. In vitro, dantrolene provides a significant protection to motor neurons exposed to a brief excitotoxic insult. However, daily administration of dantrolene to mice overexpressing superoxide dismutase 1 glycine to alanine at position 93 (SOD1G93A) does affect neither survival nor the number of motor neurons and ubiquitin aggregates indicating that calcium release through RyRs does not contribute to the selective motor neuron death in this animal model for ALS. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: neurodegeneration, endoplasmic reticulum, ryanodine receptors, motor neuron disease, amyotrophic lateral sclerosis.
INTRODUCTION Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease, which involves the loss of motor neurons and denervation of muscle fibers, *Correspondence to: L. Van Den Bosch, Campus Gasthuisberg, O&N 4 Herestraat 49 – bus 912, B-3000 Leuven, Belgium. Tel: +32-16330681; fax: +32-16-330770. E-mail address: [email protected] (L. Van Den Bosch). Abbreviations: ALS, Amyotrophic Lateral Sclerosis; AMPA, a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid; BSA, bovine serum albumin; ER, endoplasmic reticulum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HPLC, high-performance liquid chromatography; RyR, ryanodine receptor; SOD1, superoxide dismutase 1; SOD1G93A, superoxide dismutase 1, glycine to alanine at position 93; SOD1WT, wildtype superoxide dismutase 1.
0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.06.050 26
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effect of dantrolene and the superoxide dismutase 1 glycine to alanine at position 93 (SOD1G93A) mouse model of ALS to investigate the effect of RyR inhibition on the disease process and on motor neuron degeneration.
EXPERIMENTAL PROCEDURES Primary cultures Primary cultures of neurons on astrocytic feeder layers were obtained as previously described (Van Den Bosch and Robberecht, 2000). In brief, ventral spinal cords were obtained from Wistar rats at embryonic day 14 and a motor neuron enriched population was plated on pre-established glial feeder layers. These co-cultures were pretreated for 30 min with 30 lM dantrolene (or vehicle) and dantrolene was present while cells were treated with 300 lM kainate (or vehicle) on day 6 for 30 min. This procedure has been described previously (Van Den Bosch and Robberecht, 2000; Van Damme et al., 2003). Cells were counted on days 6 and 7 and values were normalized per experiment to the control (non-kainate and non-dantrolene) condition.
Animal behavior testing Mice overexpressing either human wild-type SOD1 (SOD1WT) or human mutant SOD1G93A were purchased from Jackson Laboratories (Bar Harbor, USA) and were maintained on a C57BL/6 background. Chow and water were provided ad libitum and mice were housed under standard conditions according to the guidelines of the KU Leuven. Grip strength measurements (Chatillon, Largo, USA) occurred every 5 days from 80 days of age onwards for a subset of mice at which grip strength was determined with the mice holding a bar (fore limbs only) or when placed on a small grid (all limbs). The grip strength measurements occur in units of strength (Newton) and were normalized per mouse to the average of their grip strength values obtained between days 85 and 95. When mice had become end stage their value was 0 for this test. The graph represents the average relative grip strength per group for either fore limbs only or all limbs. The hanging wire test was used on the same subset of mice to determine disease onset, to assess the ability of the mice to hold their own weight for 60 s as previously described (Teuling et al., 2008). Disease onset was subsequently used to calculate disease duration. End stage was defined as the age at which mice could no longer right themselves within 10 s when placed on their back. End stage is used as a measurement of survival and is the moment when mice are euthanized. The experimenter providing the treatment was different than the one that performed the behavioral tests and analyzed the data. All experiments were performed with the approval of the Animal Ethics Committee of KU Leuven (P020/2010).
Dantrolene administration
was performed with the StepOnePlus (Life Technologies) with TaqMan Universal PCR Master Mix (Life Technologies). The following assays were used: b-actin (4352341E, Life Technologies), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Mm99999915_g1, Life Technologies), RyR1 (Mm01175211_m1, Life Technologies), RyR2 (Mm00465877_m1, Life Technologies), RyR3 (Mm01335482_m1, Life Technologies). Relative gene expression was determined by the 2 DDct method and normalized to the average of the non-transgenic group. Graphs represent the relative gene expression as calculated by GAPDH or b-actin expression.
Drug analysis The concentration of dantrolene was determined in mouse plasma and tissue samples by high-performance liquid chromatography (HPLC) after protein precipitation with acetonitrile. Briefly, to 100 ll of plasma or 100 ll of homogenized tissue sample in 0.5% bovine serum albumin (BSA) 10 ll of the internal standard methyl dantrolene (25 lg/ml) and 200 ll of acetonitrile was added. After vortexing and centrifugation, 30 ll of the supernatant was injected onto the HPLC column packed with Hypersil BDS 5 l (250 4.6 mm I.D.) and separation performed by a mobile phase consisting of acetonitrile and potassium dihydrogen phosphate buffer 15 mM pH 3.0 (+0.05% triethylamine) (35/65, v/v) at a flow rate of 1 ml/min and UV detection at 375 nm. Calibration curves were constructed in the respective blank human plasma or 0.5% BSA for the tissue samples. Linearity in all biological media was found in the range of 0.2–10 lg/ml. The lower limit of quantification was 0.2 lg/ml, being the lowest concentration of the calibration curve with a coefficient of variation lower than 20%. Analytical recovery (%) of dantrolene and methyldantrolene was in plasma, respectively: 60.0 ± 6.5 and 64.1 ± 5.1 and in 0.5% BSA 67.7 ± 6.8 and 72.9 ± 6.0 (mean ± SD). Coefficients of variation for intra- and interday precision and accuracy were all below 15%.
Immunohistochemistry End stage or control mice were transcardially perfused with phosphate-buffered saline (PBS) and subsequently with 4% formaldehyde. Dissected spinal cords were post-fixated overnight at 4 °C and moved to 30% sucrose for an additional night. After snap freezing, tissue was sectioned by cryostat at 40 lm and stained with SMI-32 (Covance, Princeton, USA) and with polyclonal antibodies directed against ubiquitin (Dako, Denmark). Images were collected by a Zeiss Axio Imager M1 microscope (Carl Zeiss, Germany) with an AxioCam Mrc5 camera (Carl Zeiss). For motor neuron and ubiquitinated aggregate quantification 120-day-old mice treated with vehicle and dantrolene and non-transgenic mice were assessed. Motor neurons (SMI-32 immunoreactive) and aggregates (ubiquitin immunoreactive) were counted per ventral horn of multiple sections of the lumbar spinal cord (2–6 sections) per mouse (2–3 mice per group).
Statistical analysis G93A
From the age of 60 days, SOD1 mice were treated 5 days a week with vehicle (sunflower oil) or dantrolene (D1975, Sigma– Aldrich, St. Louis, USA; 5 mg/kg in suspension in sunflower oil). Both vehicle and dantrolene were administered by oral gavage by a plastic needle (Scanbur, Karlslunde, Germany; Animal Ethics Committee of KU Leuven, P115/2010).
Reverse transcriptase and quantitative PCR Isolation of mRNA from ventral spinal cord occurred by the TriPure (Roche, Basel, Switzerland) method and reverse transcriptase PCR with random hexamers (Life Technologies, Carlsbad, USA) and Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT; Invitrogen, Carlsbad, USA). Quantitative PCR
Statistical analysis and graph design were performed with Graphpad Prism (version 5.04) software. Unpaired 2-sided Student’s t-tests were used to analyze differences between 2 groups of interest. Log-rank test was used to analyze the survival data. Significance is assumed for p < 0.05. Values are shown as mean ± standard error of the mean.
RESULTS Dantrolene protects neurons from excitotoxic cell death in vitro Dantrolene inhibits the RyR and to investigate its effect on neuronal survival, we tested it on motor neurons cultured
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for 6 days on an astrocytic feeder layer. Motor neurons are exposed to 300 lM kainate for 30 min in the presence of dantrolene or vehicle. This kainate exposure leads 24 h later to a motor neuron survival of 61.8 ± 2.7% relative to the non-challenged condition (Fig. 1). Treatment with dantrolene significantly rescues survival of motor neurons to 82.2 ± 2.2% (Fig. 1). These results imply that dantrolene is protective to motor neurons in this paradigm, which is caused by calcium influx through calcium-permeable AMPA receptors (Van Den Bosch and Robberecht, 2000). Dantrolene does not affect disease in SOD1
G93A
mice
To assess a potential beneficial role for dantrolene on neuronal survival in vivo, dantrolene was administered to mice. Analysis by HPLC of dantrolene levels in plasma 30 min after a single dantrolene feeding of 5 mg/kg shows 1.44 ± 0.23 lg of dantrolene/ml plasma. As this is similar to 1.08 ± 0.26 lg of dantrolene/ml plasma described by others (Chen et al., 2008), we treated ALS mice with the same dose of dantrolene. Administration of dantrolene by forced oral feeding of SOD1G93A mice five times a week from the age of 60 days onwards does not affect disease onset as determined by hanging wire (D0.6 days, data not shown), or survival (D1.6 days, Fig. 2A). As a consequence, also the disease duration (D0.0 days, Fig. 2B) is unaltered. This is further illustrated by the comparable loss of strength in vehicle- and dantrolene-treated SOD1G93A mice (Fig. 2C, D). These data indicate that dantrolene has no effect on the disease of SOD1G93A mice. Dantrolene does not alter pathology of SOD1G93A mice To assess whether dantrolene administration alters pathology of SOD1G93A mice, we performed immunohistochemistry on the spinal cords of mice at 120 days of age (symptomatic). Decreased amounts of motor neurons are observed in the symptomatic tissues (Fig. 3A–C, quantified in 3D), as are increased ubiquitin-positive aggregates (Fig. 3D–F, quantified in 3H). No differences were observed between the symptomatic pathology of vehicle- and dantrolene-treated controls for motor neurons (Fig. 3B, C) or ubiquitin immunoreactivity (Fig. 3E, F). Analysis of merged images of SMI-32 and ubiquitin
(Fig. 3I–K) does not demonstrate any striking differences between vehicle- or dantrolene-treated 120-day-old SOD1G93A mice. Additionally, no difference is detected at end stage between vehicle- and dantrolene-treated SOD1G93A mice (data not shown). This confirms that dantrolene does not affect motor neurons in the SOD1G93A mice. RyR expression in the spinal cord in SOD1G93A mice To investigate whether the RyRs, the targets of dantrolene, are present in the spinal cord of SOD1G93A mice, we assessed the RyR gene expression in the ventral spinal cords of non-transgenic mice (200 days old), mice overexpressing human wild-type SOD1 (SOD1WT, 200 days old) and mice overexpressing human mutant SOD1G93A at different stages of the disease by quantitative PCR. The levels of gene expression of RyR type 2 (RyR2) and 3 (RyR3) are decreased at end stage SOD1G93A mice (Fig. 4B, C). This implies that RyR types 2 and 3 may be located in neurons, as neurons are lost during the disease progression of ALS. This is less clear for RyR type 1 (Fig. 4A, RyR1). In addition, the relative expression levels of all ryanodine isoforms do not differ when comparing the ventral and dorsal sides of the mouse spinal cord (Fig. 4D). This could imply that the RyRs are not preferentially expressed by ventral neurons, as this cell type is barely present in the dorsal half of the spinal cord. The pattern of RyRs in general in the spinal cord is neuronal, as detected by a pan-isoform recognizing antibody (data not shown). In conclusion, these results confirm that RyRs are present in the spinal cord of SOD1G93A mice, as also observed in motor neurons from ALS patients (Kihira et al., 2005).
DISCUSSION Calcium is an important mediator of multiple cellular processes and an excessive increase in the intracellular calcium concentration can seriously damage and eventually kill cells. In motor neurons, glutamatergic activation of calcium-permeable AMPA-type glutamate receptors is involved in the selective vulnerability of these cells in ALS (Van Den Bosch et al., 2006). Using primary motor neurons cultured on an astrocytic feeder layer, we could reproduce the selective vulnerability of motor neurons
Fig. 1. Effect of dantrolene on the survival of cultured motor neurons after an excitotoxic challenge. Dantrolene is protective for motor neurons in vitro during 30 min of exposure to 300 lM of kainate (n = 4, p = 0.002). ⁄⁄p < 0.01.
K. A. Staats et al. / Neuroscience 220 (2012) 26–31
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Fig. 2. Effect of dantrolene administration on ALS mice. (A) Daily administration of dantrolene from day 60 onwards does not increase survival of ALS mice (n = 19, 148.6 ± 2.7 days) compared to vehicle-treated ALS mice (n = 16, 150.2 ± 2.4 days; p = 0.89). The age at which a mouse is determined ‘end stage’ is used for survival analysis. (B) There is no difference between the disease duration of vehicle- (n = 3, 17.7 ± 5.4 days) and dantrolene- (n = 3, 17.7 ± 0.9 days) treated SOD1G93A mice. Disease duration is calculated by subtracting the age of onset from survival. The lack of an effect is confirmed by the similar progression of the loss of grip strength of the fore limbs (C) and all limbs (D) of vehicle- (n = 3) and dantrolene- (n = 3) treated mice, where mice pull on a bar or grid to determine muscle strength.
Fig. 3. End stage pathology of vehicle- and dantrolene-treated mice. Ventral horns of spinal cord sections of non-transgenic (n = 2; A, E and I) and vehicle-treated (n = 2; B, F and J) and dantrolene-treated (n = 3; C, G and K) SOD1G93A mice. SMI-32 was used to visualize motor neurons (A–C) by immunohistochemistry. The number of SMI-32 positive neurons is higher in the non-transgenic (A) compared to the end stage spinal cords from SOD1G93A mice (B, C) and is quantified in (D). No difference is observed between end stage spinal cords in vehicle-treated (B) and dantrolenetreated (C) SOD1G93A mice. Ubiquitin immunoreactivity in spinal cord sections shows comparable amounts of ubiquitin in the spinal cords of vehicle(E) and dantrolene-treated SOD1G93A mice (F) as quantified in (H). Non-transgenic spinal cords do not show immunoreactivity to ubiquitin. Dashed lines delineate the ventral horn. Merged analysis images of SMI-32 and ubiquitin stainings are provided in (I–K). Scale bar = 100 lm.
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Fig. 4. Ryanodine receptor isoform expression in ventral spinal cords of non-transgenic, SOD1WT and SOD1G93A mice. (A) Relative expression of RyR type 1 is unaltered at end stage (n = 6) compared to the non-transgenic control (n = 5). (B) The relative expression of RyR type 2 is significantly decreased at end stage. (C) End stage ventral spinal cords show a decreased relative expression of RyR type 3. (D) No difference is detected between ventral (n = 10) and dorsal (n = 10) spinal cords of non-transgenic mice. ns: non-significant, ⁄p < 0.05, ⁄⁄p < 0.01 (t-test between non-transgenic and end stage SOD1G93A).
by exposing them to kainate. Motor neuron death is caused by an overstimulation of the calcium-permeable AMPA receptors as motor neuron death is inhibited by selective inhibitors of this type of AMPA receptors (Van Den Bosch et al., 2000). Due to the urgent need for therapeutic interventions for motor neuron death during ALS, we studied another player involved in the calcium metabolism; the RyR, which is responsible for calcium-induced calcium release from the ER (Kimlicka and Van Petegem, 2011). In order to block calcium release from the ER, dantrolene was used, which inhibits the RyR. The advantage of dantrolene is that it is used in the clinic as a muscle relaxant to treat malignant hyperthermia caused by mutations in the RyR gene (MacLennan et al., 1990). In this study, we used primary motor neuron cultures to test the effect of dantrolene and discovered that this inhibitor of the RyRs protects motor neurons against excitotoxicity. This neuroprotection is in accordance to results obtained with organotypic cultures of lumbar spinal cord exposed to toxic concentrations of glutamate (Rothstein and Kuncl, 1995). This effect is linked to the stimulation of calciumpermeable AMPA receptors and is not observed in other cell types that lack this type of receptor (Frandsen and Schousboe, 1992). Besides an effect on survival, dantrolene inhibits calcium spikes caused by spontaneous network activity in cultured motor neurons (Jahn et al., 2006). Although dantrolene is beneficial in vitro, treating SOD1G93A mice with this drug does not increase survival. We cannot exclude that the amount of dantrolene that reaches the motor neurons is in sufficient to be protective, although we applied the treatment protocol used to treat cerebellar neurons from a positive study (Chen et al., 2008) and we obtained similar concentrations of
dantrolene in plasma. In addition, it was shown that administration of dantrolene is beneficial in rodent models of chronic neurodegeneration (Chen et al., 2008; Liu et al., 2009) and of acute neuro-injuries (Wei and Perry, 1996; Aslan et al., 2009). An alternative explanation for the lack of effect is the limited contribution of excitotoxicity in the SOD1G93A mouse model. Using different inhibitors of AMPA receptors, a maximal benefit of only 10% is observed and also the effect of riluzole in the mutant SOD1G93A mouse model is moderate (Van Den Bosch et al., 2006). Last but not the least, we cannot exclude that in vivo the relative contribution of RyRs in the calcium metabolism of motor neurons is limited. Despite the fact that we have evidence that the RNAs for the different RyRs are present in the spinal cord of SOD1G93A mice and while similar observations were made in motor neurons from ALS patients (Kihira et al., 2005), the exact importance of this calcium release channels in motor neurons is unknown.
CONCLUSIONS Despite the fact that dantrolene is able to rescue motor neuron survival in vitro, it is not able to protect motor neurons, or prolong survival of SOD1G93A mice. This indicates that calcium release by RyRs does not play a significant role in this mouse model of ALS. Acknowledgements—We thank Caroline van Heijningen for her outstanding technical support. This work was supported by Grants from the ‘‘Fund for Scientific Research Flanders’’ (FWOVlaanderen), the University of Leuven (KU Leuven) and the Belgian Government (Interuniversity Attraction Poles, programme P6/43) of the Belgian Federal Science Policy Office.
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REFERENCES Aslan A, Cemek M, Buyukokuroglu ME, Altunbas K, Bas O, Yurumez Y, Cosar M (2009) Dantrolene can reduce secondary damage after spinal cord injury. Eur Spine J 18:1442–1451. Chen X, Tang TS, Tu H, Nelson O, Pook M, Hammer R, Nukina N, Bezprozvanny I (2008) Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 3. J Neurosci 28:12713–12724. Cox LE, Ferraiuolo L, Goodall EF, Heath PR, Higginbottom A, Mortiboys H, Hollinger HC, Hartley JA, Brockington A, Burness CE, Morrison KE, Wharton SB, Grierson AJ, Ince PG, Kirby J, Shaw PJ (2010) Mutations in CHMP2B in lower motor neuron predominant amyotrophic lateral sclerosis (ALS). PLoS One 5:e9872. Frandsen A, Schousboe A (1992) Mobilization of dantrolene-sensitive intracellular calcium pools is involved in the cytotoxicity induced by quisqualate and N-methyl-D-aspartate but not by 2-amino-3(3-hydroxy-5-methylisoxazol-4-yl)propionate and kainate in cultured cerebral cortical neurons. Proc Natl Acad Sci U S A 89:2590–2594. Grosskreutz J, Van Den Bosch L, Keller BU (2010) Calcium dysregulation in amyotrophic lateral sclerosis. Cell Calcium 47:165–174. Hernandez-Fonseca K, Massieu L (2005) Disruption of endoplasmic reticulum calcium stores is involved in neuronal death induced by glycolysis inhibition in cultured hippocampal neurons. J Neurosci Res 82:196–205. Inan S, Wei H (2010) The cytoprotective effects of dantrolene: a ryanodine receptor antagonist. Anesth Analg 111:1400–1410. Jahn K, Grosskreutz J, Haastert K, Ziegler E, Schlesinger F, Grothe C, Dengler R, Bufler J (2006) Temporospatial coupling of networked synaptic activation of AMPA-type glutamate receptor channels and calcium transients in cultured motoneurons. Neuroscience 142:1019–1029. Julien JP, Kriz J (2006) Transgenic mouse models of amyotrophic lateral sclerosis. Biochim Biophys Acta 1762:1013–1024. Kihira T, Utunomiya H, Kondo T (2005) Expression of FKBP12 and ryanodine receptors (RyRs) in the spinal cord of MND patients. Amyotroph Lateral Scler Other Motor Neuron Disord 6:94–99. Kimlicka L, Van Petegem F (2011) The structural biology of ryanodine receptors. Sci China Life Sci 54:712–724. Liu J, Tang TS, Tu H, Nelson O, Herndon E, Huynh DP, Pulst SM, Bezprozvanny I (2009) Deranged calcium signaling and
31
neurodegeneration in spinocerebellar ataxia type 2. J Neurosci 29:9148–9162. Logroscino G, Traynor BJ, Hardiman O, Chio A, Mitchell D, Swingler RJ, Millul A, Benn E, Beghi E (2010) Incidence of amyotrophic lateral sclerosis in Europe. J Neurol Neurosurg Psychiatry 81:385–390. MacLennan DH, Duff C, Zorzato F, Fujii J, Phillips M, Korneluk RG, Frodis W, Britt BA, Worton RG (1990) Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia. Nature 343:559–561. Miller RG, Mitchell JD, Lyon M, Moore DH (2007) Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND). Cochrane Database Syst Rev CD001447. Rothstein JD, Kuncl RW (1995) Neuroprotective strategies in a model of chronic glutamate-mediated motor neuron toxicity. J Neurochem 65:643–651. Teuling E, van Dis V, Wulf PS, Haasdijk ED, Akhmanova A, Hoogenraad CC, Jaarsma D (2008) A novel mouse model with impaired dynein/dynactin function develops amyotrophic lateral sclerosis (ALS)-like features in motor neurons and improves lifespan in SOD1-ALS mice. Hum Mol Genet 17:2849–2862. Van Damme P, Callewaert G, Eggermont J, Robberecht W, Van Den Bosch L (2003) Chloride influx aggravates Ca2+-dependent AMPA receptor-mediated motoneuron death. J Neurosci 23:4942–4950. Van Den Bosch L, Robberecht W (2000) Different receptors mediate motor neuron death induced by short and long exposures to excitotoxicity. Brain Res Bull 53:383–388. Van Den Bosch L, Van Damme P, Bogaert E, Robberecht W (2006) The role of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis. Biochim Biophys Acta 1762:1068–1082. Van Den Bosch L, Vandenberghe W, Klaassen H, Van Houtte E, Robberecht W (2000) Ca2+-permeable AMPA receptors and selective vulnerability of motor neurons. J Neurol Sci 180:29–34. van Es MA, Van Vught PW, Blauw HM, Franke L, Saris CG, Andersen PM, Van Den Bosch L, de Jong SW, van ‘t Slot R, Birve A, Lemmens R, de Jong V, Baas F, Schelhaas HJ, Sleegers K, Van Broeckhoven C, Wokke JH, Wijmenga C, Robberecht SW, Veldink W, Ophoff RA, van den Berg LH (2007) ITPR2 as a susceptibility gene in sporadic amyotrophic lateral sclerosis: a genome-wide association study. Lancet Neurol 6:869–877. Wei H, Perry DC (1996) Dantrolene is cytoprotective in two models of neuronal cell death. J Neurochem 67:2390–2398.
(Accepted 20 June 2012) (Available online 28 June 2012)
DANTROLENE IS NEUROPROTECTIVE IN VITRO, BUT DOES NOT AFFECT SURVIVAL IN SOD1G93A MICE K. A. STAATS, a,b M. VAN RILLAER, a W. SCHEVENEELS, a,b R. VERBESSELT, c P. VAN DAMME, a,b,d W. ROBBERECHT a,b,d AND L. VAN DEN BOSCH a,b*
resulting in muscle weakness and paralysis. The disease has an annual incidence of 2.7 cases per 100,000 people in Europe (Logroscino et al., 2010) and most patients succumb to the disease within 3–5 years after onset. On average 10% of all ALS cases are familial, of which 20% are caused by mutations in superoxide dismutase 1 (SOD1). On the basis of mutations in SOD1, ALS rodent models have been generated that predictably mimic the patient disease process (Julien and Kriz, 2006). As disease progression is indistinguishable between familial and sporadic cases, common disease mechanisms are expected. One of these mechanisms is excitotoxicity, which entails the cell death of neurons by glutamatergic overstimulation. The only drug currently available for the disease, riluzole, targets this mechanism and prolongs survival of ALS patients (Miller et al., 2007). Overstimulation by glutamate opens calcium-permeable a-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and increases intracellular calcium concentrations which can become hazardous for motor neurons (Van Den Bosch et al., 2006). It is hypothesized that release of calcium from the endoplasmic reticulum (ER) may contribute to the vulnerability of motor neurons to excitotoxicity and that blocking this calcium release would be beneficial in ALS (Grosskreutz et al., 2010). A role for ER calcium in ALS has been indicated by the higher expression of the gene encoding the ER calcium channel inositol 1,4,5-trisphosphate receptor 2 (ITPR2) in total blood from ALS patients (van Es et al., 2007) and by mutations discovered in familial ALS in the charged multivesicular body protein 2b gene (CHMP2B), resulting in impaired ER calcium concentrations (Cox et al., 2010). An important calcium release channel present in the ER is the ryanodine receptor (RyR). This receptor allows calcium-induced calcium release from the ER and blocking the RyR receptor by dantrolene decreases calcium transients in cultured motor neurons (Jahn et al., 2006) and protects neurons in vitro (Rothstein and Kuncl, 1995; Wei and Perry, 1996; Hernandez-Fonseca and Massieu, 2005; Liu et al., 2009). In addition, dantrolene administration is beneficial in rodent models of a number of neurodegenerative conditions (Inan and Wei, 2010), including spinocerebellar ataxia type 2 (Liu et al., 2009) and type 3 (Chen et al., 2008), spinal cord injury (Aslan et al., 2009) and cerebral ischemia (Wei and Perry, 1996). By inhibiting the RyR with dantrolene, we investigate whether calcium entering the cell from the extracellular space can further increase the burden of calcium by the process of calcium-induced calcium release from the ER. We use primary motor neurons to confirm the protective
a
Laboratory of Neurobiology and Leuven research Institute for Neurodegenerative Disorders (LIND), KU Leuven, Belgium
b
Vesalius Research Center, VIB, Belgium
c
Center for Clinical Pharmacology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Belgium d
Department of Neurology, Gasthuisberg, Leuven, Belgium
Abstract—Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease. One of the proposed disease mechanisms is excitotoxicity, in which excessive cytosolic calcium causes neuronal death. Although most calcium may originate from the extracellular space through activation of calcium-permeable AMPA receptors, we investigated in this study the contribution of endoplasmic reticulum calcium release by blocking the ryanodine receptor (RyR) using dantrolene. In vitro, dantrolene provides a significant protection to motor neurons exposed to a brief excitotoxic insult. However, daily administration of dantrolene to mice overexpressing superoxide dismutase 1 glycine to alanine at position 93 (SOD1G93A) does affect neither survival nor the number of motor neurons and ubiquitin aggregates indicating that calcium release through RyRs does not contribute to the selective motor neuron death in this animal model for ALS. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: neurodegeneration, endoplasmic reticulum, ryanodine receptors, motor neuron disease, amyotrophic lateral sclerosis.
INTRODUCTION Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease, which involves the loss of motor neurons and denervation of muscle fibers, *Correspondence to: L. Van Den Bosch, Campus Gasthuisberg, O&N 4 Herestraat 49 – bus 912, B-3000 Leuven, Belgium. Tel: +32-16330681; fax: +32-16-330770. E-mail address: [email protected] (L. Van Den Bosch). Abbreviations: ALS, Amyotrophic Lateral Sclerosis; AMPA, a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid; BSA, bovine serum albumin; ER, endoplasmic reticulum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HPLC, high-performance liquid chromatography; RyR, ryanodine receptor; SOD1, superoxide dismutase 1; SOD1G93A, superoxide dismutase 1, glycine to alanine at position 93; SOD1WT, wildtype superoxide dismutase 1.
0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.06.050 26
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effect of dantrolene and the superoxide dismutase 1 glycine to alanine at position 93 (SOD1G93A) mouse model of ALS to investigate the effect of RyR inhibition on the disease process and on motor neuron degeneration.
EXPERIMENTAL PROCEDURES Primary cultures Primary cultures of neurons on astrocytic feeder layers were obtained as previously described (Van Den Bosch and Robberecht, 2000). In brief, ventral spinal cords were obtained from Wistar rats at embryonic day 14 and a motor neuron enriched population was plated on pre-established glial feeder layers. These co-cultures were pretreated for 30 min with 30 lM dantrolene (or vehicle) and dantrolene was present while cells were treated with 300 lM kainate (or vehicle) on day 6 for 30 min. This procedure has been described previously (Van Den Bosch and Robberecht, 2000; Van Damme et al., 2003). Cells were counted on days 6 and 7 and values were normalized per experiment to the control (non-kainate and non-dantrolene) condition.
Animal behavior testing Mice overexpressing either human wild-type SOD1 (SOD1WT) or human mutant SOD1G93A were purchased from Jackson Laboratories (Bar Harbor, USA) and were maintained on a C57BL/6 background. Chow and water were provided ad libitum and mice were housed under standard conditions according to the guidelines of the KU Leuven. Grip strength measurements (Chatillon, Largo, USA) occurred every 5 days from 80 days of age onwards for a subset of mice at which grip strength was determined with the mice holding a bar (fore limbs only) or when placed on a small grid (all limbs). The grip strength measurements occur in units of strength (Newton) and were normalized per mouse to the average of their grip strength values obtained between days 85 and 95. When mice had become end stage their value was 0 for this test. The graph represents the average relative grip strength per group for either fore limbs only or all limbs. The hanging wire test was used on the same subset of mice to determine disease onset, to assess the ability of the mice to hold their own weight for 60 s as previously described (Teuling et al., 2008). Disease onset was subsequently used to calculate disease duration. End stage was defined as the age at which mice could no longer right themselves within 10 s when placed on their back. End stage is used as a measurement of survival and is the moment when mice are euthanized. The experimenter providing the treatment was different than the one that performed the behavioral tests and analyzed the data. All experiments were performed with the approval of the Animal Ethics Committee of KU Leuven (P020/2010).
Dantrolene administration
was performed with the StepOnePlus (Life Technologies) with TaqMan Universal PCR Master Mix (Life Technologies). The following assays were used: b-actin (4352341E, Life Technologies), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Mm99999915_g1, Life Technologies), RyR1 (Mm01175211_m1, Life Technologies), RyR2 (Mm00465877_m1, Life Technologies), RyR3 (Mm01335482_m1, Life Technologies). Relative gene expression was determined by the 2 DDct method and normalized to the average of the non-transgenic group. Graphs represent the relative gene expression as calculated by GAPDH or b-actin expression.
Drug analysis The concentration of dantrolene was determined in mouse plasma and tissue samples by high-performance liquid chromatography (HPLC) after protein precipitation with acetonitrile. Briefly, to 100 ll of plasma or 100 ll of homogenized tissue sample in 0.5% bovine serum albumin (BSA) 10 ll of the internal standard methyl dantrolene (25 lg/ml) and 200 ll of acetonitrile was added. After vortexing and centrifugation, 30 ll of the supernatant was injected onto the HPLC column packed with Hypersil BDS 5 l (250 4.6 mm I.D.) and separation performed by a mobile phase consisting of acetonitrile and potassium dihydrogen phosphate buffer 15 mM pH 3.0 (+0.05% triethylamine) (35/65, v/v) at a flow rate of 1 ml/min and UV detection at 375 nm. Calibration curves were constructed in the respective blank human plasma or 0.5% BSA for the tissue samples. Linearity in all biological media was found in the range of 0.2–10 lg/ml. The lower limit of quantification was 0.2 lg/ml, being the lowest concentration of the calibration curve with a coefficient of variation lower than 20%. Analytical recovery (%) of dantrolene and methyldantrolene was in plasma, respectively: 60.0 ± 6.5 and 64.1 ± 5.1 and in 0.5% BSA 67.7 ± 6.8 and 72.9 ± 6.0 (mean ± SD). Coefficients of variation for intra- and interday precision and accuracy were all below 15%.
Immunohistochemistry End stage or control mice were transcardially perfused with phosphate-buffered saline (PBS) and subsequently with 4% formaldehyde. Dissected spinal cords were post-fixated overnight at 4 °C and moved to 30% sucrose for an additional night. After snap freezing, tissue was sectioned by cryostat at 40 lm and stained with SMI-32 (Covance, Princeton, USA) and with polyclonal antibodies directed against ubiquitin (Dako, Denmark). Images were collected by a Zeiss Axio Imager M1 microscope (Carl Zeiss, Germany) with an AxioCam Mrc5 camera (Carl Zeiss). For motor neuron and ubiquitinated aggregate quantification 120-day-old mice treated with vehicle and dantrolene and non-transgenic mice were assessed. Motor neurons (SMI-32 immunoreactive) and aggregates (ubiquitin immunoreactive) were counted per ventral horn of multiple sections of the lumbar spinal cord (2–6 sections) per mouse (2–3 mice per group).
Statistical analysis G93A
From the age of 60 days, SOD1 mice were treated 5 days a week with vehicle (sunflower oil) or dantrolene (D1975, Sigma– Aldrich, St. Louis, USA; 5 mg/kg in suspension in sunflower oil). Both vehicle and dantrolene were administered by oral gavage by a plastic needle (Scanbur, Karlslunde, Germany; Animal Ethics Committee of KU Leuven, P115/2010).
Reverse transcriptase and quantitative PCR Isolation of mRNA from ventral spinal cord occurred by the TriPure (Roche, Basel, Switzerland) method and reverse transcriptase PCR with random hexamers (Life Technologies, Carlsbad, USA) and Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT; Invitrogen, Carlsbad, USA). Quantitative PCR
Statistical analysis and graph design were performed with Graphpad Prism (version 5.04) software. Unpaired 2-sided Student’s t-tests were used to analyze differences between 2 groups of interest. Log-rank test was used to analyze the survival data. Significance is assumed for p < 0.05. Values are shown as mean ± standard error of the mean.
RESULTS Dantrolene protects neurons from excitotoxic cell death in vitro Dantrolene inhibits the RyR and to investigate its effect on neuronal survival, we tested it on motor neurons cultured
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for 6 days on an astrocytic feeder layer. Motor neurons are exposed to 300 lM kainate for 30 min in the presence of dantrolene or vehicle. This kainate exposure leads 24 h later to a motor neuron survival of 61.8 ± 2.7% relative to the non-challenged condition (Fig. 1). Treatment with dantrolene significantly rescues survival of motor neurons to 82.2 ± 2.2% (Fig. 1). These results imply that dantrolene is protective to motor neurons in this paradigm, which is caused by calcium influx through calcium-permeable AMPA receptors (Van Den Bosch and Robberecht, 2000). Dantrolene does not affect disease in SOD1
G93A
mice
To assess a potential beneficial role for dantrolene on neuronal survival in vivo, dantrolene was administered to mice. Analysis by HPLC of dantrolene levels in plasma 30 min after a single dantrolene feeding of 5 mg/kg shows 1.44 ± 0.23 lg of dantrolene/ml plasma. As this is similar to 1.08 ± 0.26 lg of dantrolene/ml plasma described by others (Chen et al., 2008), we treated ALS mice with the same dose of dantrolene. Administration of dantrolene by forced oral feeding of SOD1G93A mice five times a week from the age of 60 days onwards does not affect disease onset as determined by hanging wire (D0.6 days, data not shown), or survival (D1.6 days, Fig. 2A). As a consequence, also the disease duration (D0.0 days, Fig. 2B) is unaltered. This is further illustrated by the comparable loss of strength in vehicle- and dantrolene-treated SOD1G93A mice (Fig. 2C, D). These data indicate that dantrolene has no effect on the disease of SOD1G93A mice. Dantrolene does not alter pathology of SOD1G93A mice To assess whether dantrolene administration alters pathology of SOD1G93A mice, we performed immunohistochemistry on the spinal cords of mice at 120 days of age (symptomatic). Decreased amounts of motor neurons are observed in the symptomatic tissues (Fig. 3A–C, quantified in 3D), as are increased ubiquitin-positive aggregates (Fig. 3D–F, quantified in 3H). No differences were observed between the symptomatic pathology of vehicle- and dantrolene-treated controls for motor neurons (Fig. 3B, C) or ubiquitin immunoreactivity (Fig. 3E, F). Analysis of merged images of SMI-32 and ubiquitin
(Fig. 3I–K) does not demonstrate any striking differences between vehicle- or dantrolene-treated 120-day-old SOD1G93A mice. Additionally, no difference is detected at end stage between vehicle- and dantrolene-treated SOD1G93A mice (data not shown). This confirms that dantrolene does not affect motor neurons in the SOD1G93A mice. RyR expression in the spinal cord in SOD1G93A mice To investigate whether the RyRs, the targets of dantrolene, are present in the spinal cord of SOD1G93A mice, we assessed the RyR gene expression in the ventral spinal cords of non-transgenic mice (200 days old), mice overexpressing human wild-type SOD1 (SOD1WT, 200 days old) and mice overexpressing human mutant SOD1G93A at different stages of the disease by quantitative PCR. The levels of gene expression of RyR type 2 (RyR2) and 3 (RyR3) are decreased at end stage SOD1G93A mice (Fig. 4B, C). This implies that RyR types 2 and 3 may be located in neurons, as neurons are lost during the disease progression of ALS. This is less clear for RyR type 1 (Fig. 4A, RyR1). In addition, the relative expression levels of all ryanodine isoforms do not differ when comparing the ventral and dorsal sides of the mouse spinal cord (Fig. 4D). This could imply that the RyRs are not preferentially expressed by ventral neurons, as this cell type is barely present in the dorsal half of the spinal cord. The pattern of RyRs in general in the spinal cord is neuronal, as detected by a pan-isoform recognizing antibody (data not shown). In conclusion, these results confirm that RyRs are present in the spinal cord of SOD1G93A mice, as also observed in motor neurons from ALS patients (Kihira et al., 2005).
DISCUSSION Calcium is an important mediator of multiple cellular processes and an excessive increase in the intracellular calcium concentration can seriously damage and eventually kill cells. In motor neurons, glutamatergic activation of calcium-permeable AMPA-type glutamate receptors is involved in the selective vulnerability of these cells in ALS (Van Den Bosch et al., 2006). Using primary motor neurons cultured on an astrocytic feeder layer, we could reproduce the selective vulnerability of motor neurons
Fig. 1. Effect of dantrolene on the survival of cultured motor neurons after an excitotoxic challenge. Dantrolene is protective for motor neurons in vitro during 30 min of exposure to 300 lM of kainate (n = 4, p = 0.002). ⁄⁄p < 0.01.
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Fig. 2. Effect of dantrolene administration on ALS mice. (A) Daily administration of dantrolene from day 60 onwards does not increase survival of ALS mice (n = 19, 148.6 ± 2.7 days) compared to vehicle-treated ALS mice (n = 16, 150.2 ± 2.4 days; p = 0.89). The age at which a mouse is determined ‘end stage’ is used for survival analysis. (B) There is no difference between the disease duration of vehicle- (n = 3, 17.7 ± 5.4 days) and dantrolene- (n = 3, 17.7 ± 0.9 days) treated SOD1G93A mice. Disease duration is calculated by subtracting the age of onset from survival. The lack of an effect is confirmed by the similar progression of the loss of grip strength of the fore limbs (C) and all limbs (D) of vehicle- (n = 3) and dantrolene- (n = 3) treated mice, where mice pull on a bar or grid to determine muscle strength.
Fig. 3. End stage pathology of vehicle- and dantrolene-treated mice. Ventral horns of spinal cord sections of non-transgenic (n = 2; A, E and I) and vehicle-treated (n = 2; B, F and J) and dantrolene-treated (n = 3; C, G and K) SOD1G93A mice. SMI-32 was used to visualize motor neurons (A–C) by immunohistochemistry. The number of SMI-32 positive neurons is higher in the non-transgenic (A) compared to the end stage spinal cords from SOD1G93A mice (B, C) and is quantified in (D). No difference is observed between end stage spinal cords in vehicle-treated (B) and dantrolenetreated (C) SOD1G93A mice. Ubiquitin immunoreactivity in spinal cord sections shows comparable amounts of ubiquitin in the spinal cords of vehicle(E) and dantrolene-treated SOD1G93A mice (F) as quantified in (H). Non-transgenic spinal cords do not show immunoreactivity to ubiquitin. Dashed lines delineate the ventral horn. Merged analysis images of SMI-32 and ubiquitin stainings are provided in (I–K). Scale bar = 100 lm.
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Fig. 4. Ryanodine receptor isoform expression in ventral spinal cords of non-transgenic, SOD1WT and SOD1G93A mice. (A) Relative expression of RyR type 1 is unaltered at end stage (n = 6) compared to the non-transgenic control (n = 5). (B) The relative expression of RyR type 2 is significantly decreased at end stage. (C) End stage ventral spinal cords show a decreased relative expression of RyR type 3. (D) No difference is detected between ventral (n = 10) and dorsal (n = 10) spinal cords of non-transgenic mice. ns: non-significant, ⁄p < 0.05, ⁄⁄p < 0.01 (t-test between non-transgenic and end stage SOD1G93A).
by exposing them to kainate. Motor neuron death is caused by an overstimulation of the calcium-permeable AMPA receptors as motor neuron death is inhibited by selective inhibitors of this type of AMPA receptors (Van Den Bosch et al., 2000). Due to the urgent need for therapeutic interventions for motor neuron death during ALS, we studied another player involved in the calcium metabolism; the RyR, which is responsible for calcium-induced calcium release from the ER (Kimlicka and Van Petegem, 2011). In order to block calcium release from the ER, dantrolene was used, which inhibits the RyR. The advantage of dantrolene is that it is used in the clinic as a muscle relaxant to treat malignant hyperthermia caused by mutations in the RyR gene (MacLennan et al., 1990). In this study, we used primary motor neuron cultures to test the effect of dantrolene and discovered that this inhibitor of the RyRs protects motor neurons against excitotoxicity. This neuroprotection is in accordance to results obtained with organotypic cultures of lumbar spinal cord exposed to toxic concentrations of glutamate (Rothstein and Kuncl, 1995). This effect is linked to the stimulation of calciumpermeable AMPA receptors and is not observed in other cell types that lack this type of receptor (Frandsen and Schousboe, 1992). Besides an effect on survival, dantrolene inhibits calcium spikes caused by spontaneous network activity in cultured motor neurons (Jahn et al., 2006). Although dantrolene is beneficial in vitro, treating SOD1G93A mice with this drug does not increase survival. We cannot exclude that the amount of dantrolene that reaches the motor neurons is in sufficient to be protective, although we applied the treatment protocol used to treat cerebellar neurons from a positive study (Chen et al., 2008) and we obtained similar concentrations of
dantrolene in plasma. In addition, it was shown that administration of dantrolene is beneficial in rodent models of chronic neurodegeneration (Chen et al., 2008; Liu et al., 2009) and of acute neuro-injuries (Wei and Perry, 1996; Aslan et al., 2009). An alternative explanation for the lack of effect is the limited contribution of excitotoxicity in the SOD1G93A mouse model. Using different inhibitors of AMPA receptors, a maximal benefit of only 10% is observed and also the effect of riluzole in the mutant SOD1G93A mouse model is moderate (Van Den Bosch et al., 2006). Last but not the least, we cannot exclude that in vivo the relative contribution of RyRs in the calcium metabolism of motor neurons is limited. Despite the fact that we have evidence that the RNAs for the different RyRs are present in the spinal cord of SOD1G93A mice and while similar observations were made in motor neurons from ALS patients (Kihira et al., 2005), the exact importance of this calcium release channels in motor neurons is unknown.
CONCLUSIONS Despite the fact that dantrolene is able to rescue motor neuron survival in vitro, it is not able to protect motor neurons, or prolong survival of SOD1G93A mice. This indicates that calcium release by RyRs does not play a significant role in this mouse model of ALS. Acknowledgements—We thank Caroline van Heijningen for her outstanding technical support. This work was supported by Grants from the ‘‘Fund for Scientific Research Flanders’’ (FWOVlaanderen), the University of Leuven (KU Leuven) and the Belgian Government (Interuniversity Attraction Poles, programme P6/43) of the Belgian Federal Science Policy Office.
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REFERENCES Aslan A, Cemek M, Buyukokuroglu ME, Altunbas K, Bas O, Yurumez Y, Cosar M (2009) Dantrolene can reduce secondary damage after spinal cord injury. Eur Spine J 18:1442–1451. Chen X, Tang TS, Tu H, Nelson O, Pook M, Hammer R, Nukina N, Bezprozvanny I (2008) Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 3. J Neurosci 28:12713–12724. Cox LE, Ferraiuolo L, Goodall EF, Heath PR, Higginbottom A, Mortiboys H, Hollinger HC, Hartley JA, Brockington A, Burness CE, Morrison KE, Wharton SB, Grierson AJ, Ince PG, Kirby J, Shaw PJ (2010) Mutations in CHMP2B in lower motor neuron predominant amyotrophic lateral sclerosis (ALS). PLoS One 5:e9872. Frandsen A, Schousboe A (1992) Mobilization of dantrolene-sensitive intracellular calcium pools is involved in the cytotoxicity induced by quisqualate and N-methyl-D-aspartate but not by 2-amino-3(3-hydroxy-5-methylisoxazol-4-yl)propionate and kainate in cultured cerebral cortical neurons. Proc Natl Acad Sci U S A 89:2590–2594. Grosskreutz J, Van Den Bosch L, Keller BU (2010) Calcium dysregulation in amyotrophic lateral sclerosis. Cell Calcium 47:165–174. Hernandez-Fonseca K, Massieu L (2005) Disruption of endoplasmic reticulum calcium stores is involved in neuronal death induced by glycolysis inhibition in cultured hippocampal neurons. J Neurosci Res 82:196–205. Inan S, Wei H (2010) The cytoprotective effects of dantrolene: a ryanodine receptor antagonist. Anesth Analg 111:1400–1410. Jahn K, Grosskreutz J, Haastert K, Ziegler E, Schlesinger F, Grothe C, Dengler R, Bufler J (2006) Temporospatial coupling of networked synaptic activation of AMPA-type glutamate receptor channels and calcium transients in cultured motoneurons. Neuroscience 142:1019–1029. Julien JP, Kriz J (2006) Transgenic mouse models of amyotrophic lateral sclerosis. Biochim Biophys Acta 1762:1013–1024. Kihira T, Utunomiya H, Kondo T (2005) Expression of FKBP12 and ryanodine receptors (RyRs) in the spinal cord of MND patients. Amyotroph Lateral Scler Other Motor Neuron Disord 6:94–99. Kimlicka L, Van Petegem F (2011) The structural biology of ryanodine receptors. Sci China Life Sci 54:712–724. Liu J, Tang TS, Tu H, Nelson O, Herndon E, Huynh DP, Pulst SM, Bezprozvanny I (2009) Deranged calcium signaling and
31
neurodegeneration in spinocerebellar ataxia type 2. J Neurosci 29:9148–9162. Logroscino G, Traynor BJ, Hardiman O, Chio A, Mitchell D, Swingler RJ, Millul A, Benn E, Beghi E (2010) Incidence of amyotrophic lateral sclerosis in Europe. J Neurol Neurosurg Psychiatry 81:385–390. MacLennan DH, Duff C, Zorzato F, Fujii J, Phillips M, Korneluk RG, Frodis W, Britt BA, Worton RG (1990) Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia. Nature 343:559–561. Miller RG, Mitchell JD, Lyon M, Moore DH (2007) Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND). Cochrane Database Syst Rev CD001447. Rothstein JD, Kuncl RW (1995) Neuroprotective strategies in a model of chronic glutamate-mediated motor neuron toxicity. J Neurochem 65:643–651. Teuling E, van Dis V, Wulf PS, Haasdijk ED, Akhmanova A, Hoogenraad CC, Jaarsma D (2008) A novel mouse model with impaired dynein/dynactin function develops amyotrophic lateral sclerosis (ALS)-like features in motor neurons and improves lifespan in SOD1-ALS mice. Hum Mol Genet 17:2849–2862. Van Damme P, Callewaert G, Eggermont J, Robberecht W, Van Den Bosch L (2003) Chloride influx aggravates Ca2+-dependent AMPA receptor-mediated motoneuron death. J Neurosci 23:4942–4950. Van Den Bosch L, Robberecht W (2000) Different receptors mediate motor neuron death induced by short and long exposures to excitotoxicity. Brain Res Bull 53:383–388. Van Den Bosch L, Van Damme P, Bogaert E, Robberecht W (2006) The role of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis. Biochim Biophys Acta 1762:1068–1082. Van Den Bosch L, Vandenberghe W, Klaassen H, Van Houtte E, Robberecht W (2000) Ca2+-permeable AMPA receptors and selective vulnerability of motor neurons. J Neurol Sci 180:29–34. van Es MA, Van Vught PW, Blauw HM, Franke L, Saris CG, Andersen PM, Van Den Bosch L, de Jong SW, van ‘t Slot R, Birve A, Lemmens R, de Jong V, Baas F, Schelhaas HJ, Sleegers K, Van Broeckhoven C, Wokke JH, Wijmenga C, Robberecht SW, Veldink W, Ophoff RA, van den Berg LH (2007) ITPR2 as a susceptibility gene in sporadic amyotrophic lateral sclerosis: a genome-wide association study. Lancet Neurol 6:869–877. Wei H, Perry DC (1996) Dantrolene is cytoprotective in two models of neuronal cell death. J Neurochem 67:2390–2398.
(Accepted 20 June 2012) (Available online 28 June 2012)