Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by a substantial loss of motor neurons in the spinal cord, brainstem and motor cortex. The degeneration of motor neurons leads to skeletal muscle atrophy, paralysis and death. The mechanisms by which mutations in SOD1 lead to motor neuron degeneration remain unidentified. Several mechanisms have been suggested by which a mutation of SOD1 may lead to motor neuron toxicity and degeneration, including the formation of intracellular aggregates (Johnston et al., 2000), production of reactive oxidative species (Estevez et al., 1999), mitochondrial degeneration (Higgins et al., 2002), altered AMPA receptor permeability and subunit composition (Pieri et al., 2003; Spalloni et al., 2004), increase in intracellular Ca2+ concentration by Ca2+-permeable AMPA receptor channels (Van Den Bosch et al., 2000; Weiss et al., 2000) and protein nitration (Casoni et al., 2005). In the present work, the excitability of motor neurons and the physiological properties of the macroscopic voltage-dependent Na+, Ca++ and K+ currents have been tested in a transgenic mouse model of a familial form of ALS, associated with a mutation in Cu,Zn superoxide dismutase (Gly93 Ala). The results indicate that the passive membrane properties were not modified in G93A motor neurons compared to control neurons whereas the firing properties of single motor neurons in G93A are altered to induce in these neurons hyperexcitability. In addition, the voltage dependence of activation and of steady-state inactivation, the kinetics of fast inactivation and slow inactivation of the voltage-dependent Na+ channels were not modified in the mutated mice. Conversely, the recovery from fast inactivation was significantly faster in G93A motor neurons compared to control neurons. The recovery from fast inactivation was still significantly faster in G93A motor neurons exposed for different times (3-48 hours) and concentration (5-500 μM) to edaravone, a free radical scavenger. The analysis of the voltage-dependent calcium currents and potassium currents showed not significant differences in all the parameters studied in the three neuronal populations. Finally, to investigate which modifications at the ionic current level can produce the observed hyperexcitability in G93A motor neurons, we developed a numerical simulator of the electrical activity of mouse spinal motor neuron. We found that changes limited to ionic current conductances or to faster recovery from fast inactivation of the Na+ channels are not able to reproduce G93A firing alterations. We observed that the mutant motor neuron hyperexcitability can be reproduced by means of a faster kinetic of small conductance calcium-dependent potassium current. These results indicate for the first time that, in accordance with clinical data, the firing properties of single motor neurons in a genetic mouse model of ALS are altered to induce neuronal hyperexcitability and that they may contribute to the pathogenesis of the disease. The clarification of the importance of these changes in membrane ion channel functionality may have diagnostic and therapeutic implications in the pathogenesis of ALS. This alteration, demonstrated at the single-cell level, may affect the normal orchestrated activation and inactivation gating of the voltagedependent channels involved in the neuronal excitability and may be significant in the onset and progression of the pathology. For these reasons, an electrophysiological characterization of small conductance calcium-activated potassium current will be necessary in order to experimentally verify the simulation results.

Pieri, M. (2009). Alterazione funzionale dei canali ionici nel sistema nervoso centrale in un modello murino di sclerosi laterale amiotrofica.

Alterazione funzionale dei canali ionici nel sistema nervoso centrale in un modello murino di sclerosi laterale amiotrofica

PIERI, MASSIMO
2009-07-14

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by a substantial loss of motor neurons in the spinal cord, brainstem and motor cortex. The degeneration of motor neurons leads to skeletal muscle atrophy, paralysis and death. The mechanisms by which mutations in SOD1 lead to motor neuron degeneration remain unidentified. Several mechanisms have been suggested by which a mutation of SOD1 may lead to motor neuron toxicity and degeneration, including the formation of intracellular aggregates (Johnston et al., 2000), production of reactive oxidative species (Estevez et al., 1999), mitochondrial degeneration (Higgins et al., 2002), altered AMPA receptor permeability and subunit composition (Pieri et al., 2003; Spalloni et al., 2004), increase in intracellular Ca2+ concentration by Ca2+-permeable AMPA receptor channels (Van Den Bosch et al., 2000; Weiss et al., 2000) and protein nitration (Casoni et al., 2005). In the present work, the excitability of motor neurons and the physiological properties of the macroscopic voltage-dependent Na+, Ca++ and K+ currents have been tested in a transgenic mouse model of a familial form of ALS, associated with a mutation in Cu,Zn superoxide dismutase (Gly93 Ala). The results indicate that the passive membrane properties were not modified in G93A motor neurons compared to control neurons whereas the firing properties of single motor neurons in G93A are altered to induce in these neurons hyperexcitability. In addition, the voltage dependence of activation and of steady-state inactivation, the kinetics of fast inactivation and slow inactivation of the voltage-dependent Na+ channels were not modified in the mutated mice. Conversely, the recovery from fast inactivation was significantly faster in G93A motor neurons compared to control neurons. The recovery from fast inactivation was still significantly faster in G93A motor neurons exposed for different times (3-48 hours) and concentration (5-500 μM) to edaravone, a free radical scavenger. The analysis of the voltage-dependent calcium currents and potassium currents showed not significant differences in all the parameters studied in the three neuronal populations. Finally, to investigate which modifications at the ionic current level can produce the observed hyperexcitability in G93A motor neurons, we developed a numerical simulator of the electrical activity of mouse spinal motor neuron. We found that changes limited to ionic current conductances or to faster recovery from fast inactivation of the Na+ channels are not able to reproduce G93A firing alterations. We observed that the mutant motor neuron hyperexcitability can be reproduced by means of a faster kinetic of small conductance calcium-dependent potassium current. These results indicate for the first time that, in accordance with clinical data, the firing properties of single motor neurons in a genetic mouse model of ALS are altered to induce neuronal hyperexcitability and that they may contribute to the pathogenesis of the disease. The clarification of the importance of these changes in membrane ion channel functionality may have diagnostic and therapeutic implications in the pathogenesis of ALS. This alteration, demonstrated at the single-cell level, may affect the normal orchestrated activation and inactivation gating of the voltagedependent channels involved in the neuronal excitability and may be significant in the onset and progression of the pathology. For these reasons, an electrophysiological characterization of small conductance calcium-activated potassium current will be necessary in order to experimentally verify the simulation results.
A.A. 2005/2006
Neuroscienze
19.
ASL; action potentials; motor neurons; sodium current
Settore MED/26 - Neurologia
Italian
Tesi di dottorato
Pieri, M. (2009). Alterazione funzionale dei canali ionici nel sistema nervoso centrale in un modello murino di sclerosi laterale amiotrofica.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2108/927
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