An α-mercaptoacrylic acid derivative (PD150606) inhibits selective motor neuron death via inhibition of kainate-induced Ca2+ influx and not via calpain inhibition

Neurobiology, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.
Neuropharmacology (Impact Factor: 5.11). 05/2002; 42(5):706-13. DOI: 10.1016/S0028-3908(02)00010-2
Source: PubMed


Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neuron death. The exact mechanism responsible for this selectivity is not clear, although it is known that motor neurons are very sensitive to excitotoxicity. This high sensitivity is due to a high density of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors on their surface and to a limited Ca(2+) buffering capacity. Ca(2+) can enter the cell upon stimulation through voltage-operated Ca(2+) channels and through the Ca(2+)-permeable portion of AMPA receptors. How this Ca(2+) kills motor neurons is incompletely understood. In the present study, we report that kainate (KA)-induced motor neuron death is purely mediated through Ca(2+) entering motor neurons through Ca(2+)-permeable AMPA receptors and that voltage-operated Ca(2+) channels play no significant role. In contrast to what has been observed in other neuronal models or after N-methyl-D-aspartate stimulation, NO synthase inhibition and a number of antioxidants did not protect motor neurons from KA-induced death. Only PD150606, derived from alpha-mercaptoacrylic acid and considered as a selective calpain antagonist, inhibited dose-dependently the KA-induced motor neuron death. However, other calmodulin and calpain inhibitors were not effective. At least part of the inhibitory effect of PD150606 is due to an irreversible inhibition of the Ca(2+) influx through the Ca(2+)-permeable AMPA receptor. These results demonstrate the interesting property of PD150606 to interfere with excitotoxicity-dependent motor neuron death and show that PD150606 is not an exclusive calpain/calmodulin antagonist.

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Available from: Ludo Van Den Bosch, Jan 04, 2014
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    • "As with calpeptin, 10 μM PD150606 did not decrease the incidence of DCD relative to vehicle treated cultures after 60 min of glutamate treatment although the incidence was slightly reduced at 20 min (only significant for 20% O 2 cultures, Table 1). However, increasing PD150606 to 50 μM, a concentration not specific for calpains (Wang et al. 1996;Van den et al. 2002), resulted in significant protection of neurons at both 20 and 60 min (Table 1). "
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    ABSTRACT: Although calpain (EC 3.4.22) protease activation was suggested to contribute to excitotoxic delayed calcium deregulation (DCD) via proteolysis of Na+/Ca2+ exchanger 3 (NCX3), cytoplasmic calpain activation in relation to DCD has never been visualized in real-time. We employed a calpain fluorescence resonance energy transfer substrate to simultaneously image calpain activation and calcium deregulation in live cortical neurons. A calpain inhibitor-sensitive decline in fluorescence resonance energy transfer was observed at 39 +/- 5 min after the occurrence of DCD in neurons exposed to continuous glutamate (100 microM). Inhibition of calpain by calpeptin did not delay the onset of DCD, recovery from DCD-like reversible calcium elevations, or cell death despite inhibiting alpha-spectrin processing by > 90%. NCXs reversed during glutamate exposure, the NCX antagonist KB-R7943 prolonged the time to DCD, and significant NCX3 cleavage following 90 min of glutamate exposure was not observed. Our findings suggest that robust calpain activation associated with acute glutamate toxicity occurs only after a sustained loss in calcium homeostasis. Processing of NCX3 or other calpain substrates is unlikely to be the primary cause of acute excitotoxicity in cortical neurons. However, a role for calpain as a contributing factor or in response to milder glutamate insults is not excluded.
    Journal of Neurochemistry 05/2009; 110(3):990-1004. DOI:10.1111/j.1471-4159.2009.06194.x · 4.28 Impact Factor
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    • "Several series of AMPA receptor antagonists are reported in the literature including AMPA-derived antagonists as well as the quinoxalinedione derivatives (Madsen et al., 2001). Antagonists of AMPA receptors have been shown to be consistently effective in reducing neuronal death in various experimental models (Canton et al., 2001; Van Damme et al., 2003; Van den Bosch et al., 2002). However, their full potential has not been realized in clinical applications due to their severe side effects that can be mainly attributed to their insolubility (Herrling, 1997; Sheardown et al., 1990). "
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    ABSTRACT: Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are one of the important receptor classes involved in glutamate-mediated excitotoxicity. Although small molecule antagonists of this receptor have been shown to have neuroprotective properties, their low solubilities pose severe side effects in clinical trials. Here we have used the SELEX method to obtain water-soluble nuclease-resistant RNA ligands that bind to the agonist binding site of AMPA receptors. Using whole-cell current recordings, we have characterized the functional consequences of a representative aptamer from this class and show that it is a competitive antagonist of AMPA receptors and in the concentration range where it acts as an inhibitor of the AMPA receptor the RNA has no effect on the GluR6 homomeric kainate receptors. Additionally, using a fluorescence resonance energy transfer (FRET) probe, we show that this RNA ligand stabilizes the open cleft conformation of the ligand binding domain, consistent with the known structures of small antagonist-bound states of the soluble domain of this protein. Finally, using rat primary cortical neurons, we show that this RNA ligand significantly reduces neurotoxicity associated with oxygen glucose deprivation. The water-soluble and antagonistic properties of this aptamer coupled with its neuroprotective properties make it an excellent candidate for potential use in diseases or pathological conditions involving glutamate-mediated excitotoxicity.
    Neuropharmacology 09/2007; 53(2):242-51. DOI:10.1016/j.neuropharm.2007.05.007 · 5.11 Impact Factor
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    • "We have shown that NBQX, an AMPA receptor antagonist, prevents kainate-induced motor neuron death in this in vitro system and also prolongs survival in a transgenic mouse model for ALS (Van Damme et al., 2003b). We also used these motor neuron cultures to study the post-receptor mechanisms leading to Ca 2+ permeable AMPA receptor-dependent neuronal injury, but could not find an effect of inhibitors of the Ca 2+ -activated enzyme nitric oxide synthase, neither of inhibitors of calmodulin or calpain, nor of antioxidants (Van Den Bosch et al., 2002). "
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    ABSTRACT: alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated excitotoxicity contributes to the selective motor neuron death in amyotrophic lateral sclerosis (ALS). In this study, we investigated the effect of P2 receptor-influencing substances on kainate-induced motor neuron death in an in vitro model for AMPA receptor-mediated excitotoxicity. Complete protection was found after preincubation of the motor neurons with ivermectin or Cibacron Blue 3G-A. Preincubation with both P2X4 modulators did not influence the number or Ca2+ permeability of the AMPA receptors and addition during kainate stimulation alone had no effect. Preincubation with a low concentration of ATP, the natural agonist of the P2X4 receptor, also protected the motor neurons against a subsequent excitotoxic stimulation, while high concentrations of ATP were toxic. Moreover, ivermectin increased the toxicity of low ATP concentrations, indicating that ivermectin can potentiate the effect of ATP on its receptor. Ivermectin and ATP also protected against hypoxia/hypoglycemia. To further investigate the relevance of these findings for ALS, we treated SOD1(G93A)-mice, a transgenic animal model for familial ALS, with ivermectin. This resulted in an extension of the life span of these mice with almost 10%. We conclude that ivermectin induces a mechanism in motor neurons, in vivo and in vitro, that protects against subsequent excitotoxic insults. Our in vitro data indicate that this protective mechanism is due to the potentiation by ivermectin of an effect of ATP mediated by the P2X4 receptor.
    Neurobiology of Disease 02/2007; 25(1):8-16. DOI:10.1016/j.nbd.2006.08.018 · 5.08 Impact Factor
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