ABSTRACT
Title
Abnormal glutamate exocytosis: a possible cause of excyotoxicy in ALS
Authors
M. Milanese1, T. Bonifacino1, F. Onofri1, L. Musazzi2, F. Giribaldi1, A. Puliti3, Fabio Benfenati1,4, M. Popoli2,5, G. Bonanno1,6
1Dept. Exp. Med., Univ. of Genoa, Italy;
2Dept. Pharmacol. Sci. Univ. of Milan, Italy;
2Dept. of Ped. Sci., Univ. of Genoa and Mol. Genet. and Cytogenet. Unit, Gaslini Inst. Genoa, Italy;
4Italian Inst. Technol., Genoa, Italy;
5Ctr. of Excellence for Neurodegenerative Dis., Univ. of Milan, Italy;
6Ctr. of Excellence for Biomed. Res., Univ. of Genoa, Italy.
1Dept. Exp. Med., Univ. of Genoa, Italy;
2Dept. Pharmacol. Sci. Univ. of Milan, Italy;
2Dept. of Ped. Sci., Univ. of Genoa and Mol. Genet. and Cytogenet. Unit, Gaslini Inst. Genoa, Italy;
4Italian Inst. Technol., Genoa, Italy;
5Ctr. of Excellence for Neurodegenerative Dis., Univ. of Milan, Italy;
6Ctr. of Excellence for Biomed. Res., Univ. of Genoa, Italy.
Abstract
Amyotrophic lateral sclerosis (ALS) is a late-onset fatal neurodegenerative disease characterized by muscle wasting, weakness and spasticity, reflecting a degeneration of upper and lower motor-neurons. The cause of ALS and the mechanisms of neuronal death are still largely obscure, thus impairing the establishment of efficacious therapies. Glutamate(Glu)-mediated excitotoxicity plays a major role in the degeneration of motor neurons in ALS and reduced astrocytary glutamate transport, which in turn increases the synaptic availability of the amino acid neurotransmitter, was suggested as a cause (1). On the basis of our previous studies (1, 2), we alternatively proposed that abnormal release of Glu may well represent a possible source of excessive Glu transmission.
Here we demonstrate that the neuronal release of Glu induced by depolarizing and non depolarizing stimuli or by Group 1 metabotropic glutamate receptor (mGLu1 and mGlu5) activation, known to induce exocytotic neurotransmitter release, is abnormal in mice expressing human SOD1 with the G93A substitution (SOD1/G93A), a transgenic model of ALS, respect to mice expressing the unmodified human SOD1 (SOD1) and to non transgenic littermates.
Exposure to 15 or 25 mM KCl or to 0.3 or 1 µM ionomycin provoked an almost complete Ca2+-dependent release of Glu from spinal cord synaptosomes. This exocytotic release was dramatically increased in late-symptomatic, early-symptomatic and pre-symptomatic SOD1/G93A mice respect to controls. Noticeably, the stimulus-evoked release of GABA or Glycine in spinal cord of SOD1/G93A mice did not differ from controls. Increased Ca2+ levels were detected in SOD1/G93A mouse spinal cord nerve terminals, accompanied by increased activation of Ca2+/calmodulin-dependent kinase II and increased phosphorylation of synapsin I. In line with these findings, release experiments suggested that the glutamate release augmentation in SOD1/G93A mice involves the readily releasable pool of vesicles and a greater capability of these vesicles to fusion upon stimulation.
mGlu1 and mGlu5 Glu-releasing autoreceptors induce Glu release in cerebral cortex nerve terminals (4). We here demonstrate the existence of the same receptors in mouse spinal cord, whose activation produces higher Glu release in SOD1/G93A mice than in controls. In fact, the mixed mGlu1/5 receptor agonist DHPG, potentiated the release of Glu from SOD1/G93A and control mice by a similar extent, at concentrations ≥ 3 µM. At variance, activation of mGlu receptors by <1 µM DHPG stimulated Glu release in SOD1/G93A mice only, with the agonist inactive in control mice. Antagonism of DHPG effect by MPEP or fenobam (mGlu5 receptors) or by CPCOOET or LY367385 (mGlu1 receptors) evidenced that the high affinity effect is supported by mGlu5 and the low affinity by mGlu1 receptor activation. Accordingly, mGlu5 receptors were over-expressed in SOD1/G93A mice. Moreover, 0.3 µM DHPG stimulated IP3 production in SOD1/G93A mice only, whereas 30 µM DHPG stimulated IP3 in both SOD1/G93A and SOD1 mice. DHPG effects were almost insensitive to omission of external Ca2+, abolished in the presence of BAPTA or of the IP3 receptor blocker 2-APB, suggesting internal Ca2+ mobilization via IP3-sensitive channels.
Our in-vitro results show that the exocytotic release of Glu is enhanced in mutant SOD1 mice under different experimental conditions. If it occurs in-vivo, the anomalous modulation of Glu release could induce an unbalance between spinal inhibitory and excitatory transmission in ALS. In line, ongoing experiments are depicting a role in-vivo for Group 1 metabotropic Glu receptors in the development of the pathology.
Supported by MURST (PRIN project 2006058401) and by Italian Ministry of Health (Ricerca Finalizzata)
(1) Rothstein et al, Ann Neurol 38: 73, 1995
(2) Raiteri et al, Neuropharmacology 46: 782, 2004
(3) Milanese et al, J Neurochem 113: 489, 2010
(4) Musante et al, Neuropharmacology 55: 474, 2008
Here we demonstrate that the neuronal release of Glu induced by depolarizing and non depolarizing stimuli or by Group 1 metabotropic glutamate receptor (mGLu1 and mGlu5) activation, known to induce exocytotic neurotransmitter release, is abnormal in mice expressing human SOD1 with the G93A substitution (SOD1/G93A), a transgenic model of ALS, respect to mice expressing the unmodified human SOD1 (SOD1) and to non transgenic littermates.
Exposure to 15 or 25 mM KCl or to 0.3 or 1 µM ionomycin provoked an almost complete Ca2+-dependent release of Glu from spinal cord synaptosomes. This exocytotic release was dramatically increased in late-symptomatic, early-symptomatic and pre-symptomatic SOD1/G93A mice respect to controls. Noticeably, the stimulus-evoked release of GABA or Glycine in spinal cord of SOD1/G93A mice did not differ from controls. Increased Ca2+ levels were detected in SOD1/G93A mouse spinal cord nerve terminals, accompanied by increased activation of Ca2+/calmodulin-dependent kinase II and increased phosphorylation of synapsin I. In line with these findings, release experiments suggested that the glutamate release augmentation in SOD1/G93A mice involves the readily releasable pool of vesicles and a greater capability of these vesicles to fusion upon stimulation.
mGlu1 and mGlu5 Glu-releasing autoreceptors induce Glu release in cerebral cortex nerve terminals (4). We here demonstrate the existence of the same receptors in mouse spinal cord, whose activation produces higher Glu release in SOD1/G93A mice than in controls. In fact, the mixed mGlu1/5 receptor agonist DHPG, potentiated the release of Glu from SOD1/G93A and control mice by a similar extent, at concentrations ≥ 3 µM. At variance, activation of mGlu receptors by <1 µM DHPG stimulated Glu release in SOD1/G93A mice only, with the agonist inactive in control mice. Antagonism of DHPG effect by MPEP or fenobam (mGlu5 receptors) or by CPCOOET or LY367385 (mGlu1 receptors) evidenced that the high affinity effect is supported by mGlu5 and the low affinity by mGlu1 receptor activation. Accordingly, mGlu5 receptors were over-expressed in SOD1/G93A mice. Moreover, 0.3 µM DHPG stimulated IP3 production in SOD1/G93A mice only, whereas 30 µM DHPG stimulated IP3 in both SOD1/G93A and SOD1 mice. DHPG effects were almost insensitive to omission of external Ca2+, abolished in the presence of BAPTA or of the IP3 receptor blocker 2-APB, suggesting internal Ca2+ mobilization via IP3-sensitive channels.
Our in-vitro results show that the exocytotic release of Glu is enhanced in mutant SOD1 mice under different experimental conditions. If it occurs in-vivo, the anomalous modulation of Glu release could induce an unbalance between spinal inhibitory and excitatory transmission in ALS. In line, ongoing experiments are depicting a role in-vivo for Group 1 metabotropic Glu receptors in the development of the pathology.
Supported by MURST (PRIN project 2006058401) and by Italian Ministry of Health (Ricerca Finalizzata)
(1) Rothstein et al, Ann Neurol 38: 73, 1995
(2) Raiteri et al, Neuropharmacology 46: 782, 2004
(3) Milanese et al, J Neurochem 113: 489, 2010
(4) Musante et al, Neuropharmacology 55: 474, 2008