ABSTRACT
Title
Excessive and precocious glutamate release in the spinal cord of a mouse model of amyotrophic lateral sclerosis
Authors
T. Bonifacino1, M. Milanese1, F. Onofri2, L. Musazzi 3, D. Tardito3, M. Messa4, C. Usai5, G. Racagni3,6, F. Benfenati2,7, M. Popoli3, G. Bonanno1.
1Dept. of Experimental Medicine, Pharmacology and Toxicology Section and Centre of Excellence for Biomedical Research, University of Genoa, Italy.
2Dept. of Experimental Medicine, Section of Physiology, University of Genoa - Italy.
3Center of Neuropharmacology - Dept. of Pharmacological Sciences and Center of Excellence on
Neurodegenerative Diseases, University of Milan - Italy.
4Department of Cell Biology, Yale School of Medicine, New Haven CT USA.
5Institute of Biophysics, National Research Council, Genova, Italy
6 I.R.C.C.S. San Giovanni di Dio - Fatebenefratelli, Brescia - Italy.
7Dept. of Neuroscience and Brain Technologies, The Italian Institute of Technology, Genoa - Italy.
1Dept. of Experimental Medicine, Pharmacology and Toxicology Section and Centre of Excellence for Biomedical Research, University of Genoa, Italy.
2Dept. of Experimental Medicine, Section of Physiology, University of Genoa - Italy.
3Center of Neuropharmacology - Dept. of Pharmacological Sciences and Center of Excellence on
Neurodegenerative Diseases, University of Milan - Italy.
4Department of Cell Biology, Yale School of Medicine, New Haven CT USA.
5Institute of Biophysics, National Research Council, Genova, Italy
6 I.R.C.C.S. San Giovanni di Dio - Fatebenefratelli, Brescia - Italy.
7Dept. of Neuroscience and Brain Technologies, The Italian Institute of Technology, Genoa - Italy.
Abstract
Amyotrophic lateral sclerosis (ALS) is a chronic, neuromuscular disorder characterized by muscle wasting, weakness, and spasticity, reflecting a progressive neurodegeneration of upper and lower motor-neurons. Selective vulnerability of motor-neurons has been in turn ascribed as a consequence of multifactorial causes: protein misfolding, mitochondrial dysfunction, oxidative damage, insufficient growth factor signaling, inflammation and glutamate(Glu)-mediated excitotoxicity. High glutamate levels have been reported in ALS patients as well as in animal models of the disease, moreover a reduced glutamate transport was suggested as a cause.
Due to the complex interplay of multiple mechanisms in the aetiology of ALS, defects of glutamate transport may not be the only reason for excitotoxicity-based neurodegeneration and other causes should be considered for the augmented glutamate availability, including increase of glutamate release.We here studied the release of [3H]D-aspartate and endogenous glutamate, induced by exocytotic stimuli in synaptosomes obtained from spinal cord of mice expressing human SOD1 with the G93A mutation [SOD1/G93A(+)], a transgenic model of familial ALS,as a potential cause for the hyper-glutamatergicity.
The spontaneous outflow of glutamate was more elevated in SOD1/G93A(+) mice, as compared to mice expressing wild type human SOD1 or to non-transgenic controls. Exposure to 15 mMKCl or 0.3 µM ionomycin provoked Ca2+-dependent glutamate release that was dramatically increased in symptomatic transgenic mutated mice. Contrary to glutamate, the stimulus-evoked release of [3H]GABA or [3H]glycine in the spinal cord of SOD1/G93A(+) mice did not differ from controls, and the same was true for [3H]D-aspartate release in the motor-cortex. The augmentation of basal and stimulated glutamate release was already present in pre-symptomatic mutant mice. Further studies revealed the existence of increased resting and stimulated Ca2+ levels in nerve terminals from the G93A mice spinal cord, accompanied by increased activation of Ca2+/calmodulin-dependent kinase II and increased phosphorylation of synapsyn-I. In line with this findings, release experiments suggested the involvement of the readily releasable pool of vesicles and a greater capability of these vesicles to fuse upon stimulation.
We can conclude that Glu exocytosis is elevated in symptomatic and pre-symptomatic G93A mutant mice and that changes in cytosolic Ca2+ concentrations, Ca2+/calmodulin-dependent protein kinase II (CaMKII) auto-activation and synapsin I phosphorylation seem to be the major causes of the augmented excitatory neurotransmitter release.
Due to the complex interplay of multiple mechanisms in the aetiology of ALS, defects of glutamate transport may not be the only reason for excitotoxicity-based neurodegeneration and other causes should be considered for the augmented glutamate availability, including increase of glutamate release.We here studied the release of [3H]D-aspartate and endogenous glutamate, induced by exocytotic stimuli in synaptosomes obtained from spinal cord of mice expressing human SOD1 with the G93A mutation [SOD1/G93A(+)], a transgenic model of familial ALS,as a potential cause for the hyper-glutamatergicity.
The spontaneous outflow of glutamate was more elevated in SOD1/G93A(+) mice, as compared to mice expressing wild type human SOD1 or to non-transgenic controls. Exposure to 15 mMKCl or 0.3 µM ionomycin provoked Ca2+-dependent glutamate release that was dramatically increased in symptomatic transgenic mutated mice. Contrary to glutamate, the stimulus-evoked release of [3H]GABA or [3H]glycine in the spinal cord of SOD1/G93A(+) mice did not differ from controls, and the same was true for [3H]D-aspartate release in the motor-cortex. The augmentation of basal and stimulated glutamate release was already present in pre-symptomatic mutant mice. Further studies revealed the existence of increased resting and stimulated Ca2+ levels in nerve terminals from the G93A mice spinal cord, accompanied by increased activation of Ca2+/calmodulin-dependent kinase II and increased phosphorylation of synapsyn-I. In line with this findings, release experiments suggested the involvement of the readily releasable pool of vesicles and a greater capability of these vesicles to fuse upon stimulation.
We can conclude that Glu exocytosis is elevated in symptomatic and pre-symptomatic G93A mutant mice and that changes in cytosolic Ca2+ concentrations, Ca2+/calmodulin-dependent protein kinase II (CaMKII) auto-activation and synapsin I phosphorylation seem to be the major causes of the augmented excitatory neurotransmitter release.