Voltage gated sodium channels (VGSCs) are multimeric protein complexes formed by one alpha subunit, responsible of pore formation and of the regulation of the state of the channel, and by one or two accessory beta subunits that have an important role in the regulation of alpha subunit localization and kinetics and in the interaction with the extracellular environment. VGSCs, together with Calcium and Potassium voltage dependent channels, are involved in the definition of cellular electrical properties. In particular, a small percentage (1%) of the voltage dependent Na+ current generated by VGSCs is a non-inactivating component (Persistent Sodium Current, INaP). Recent evidence pointed out an involvement of VGSCs and INaP in promoting neurite elongation in health and disease. Indeed, NaV 1.6 and b1 subunits were reported to be fundamental in axonal outgrowth in mouse neuron primary culture. Moreover, our previous studies on a zebrafish model of Amyotrophic Lateral Sclerosis, that express the mutant G93R form of the superoxide dismutase 1 (SOD1) enzyme, show that alteration in the INaP of spinal inter- and moto- neurons induced neuron hyperexcitability that finally leads to hyperactive zebrafish locomotor behaviour together with morphological alterations of motoneuronal phenotype. In this scenario, we decided to deeper investigate the role of VGSCs in neurite elongation, by taking advantage of the NSC34 cell line, an in vitro model often used in the study of motoneuron disease. Thus, we used two different NSC34 model of ALS, the first one expressing the human wild-type (WT) or G93A form of the SOD1 enzyme and the second one that lacks for the expression of the vesicle-associated membrane protein associated protein B (VAPB) protein. By mean of different experimental approaches we study the expression of VGSCs protein and mRNA levels, the localization of the channel and the ability of the cell to respond to a depolarizing stimulus in health and disease in vitro models. Then, taking advantage of pharmacological approaches we evaluate whether changes in VGSCs functionality cause impairments in neurites elongation. Surprisingly, we found a strong correlation between VGSCs expression, localization and functionality and neuron differentiation in all the model we analysed. In particular, VGSCs increased expression seems to be functional to the increase in the percentage of differentiated cells during NSC34 differentiation. Moreover, NSC34 defective neurite outgrowth correlates with VGSCs defective expression or localization in NSC34 ALS models. In conclusion, the study that I have conduct during the three years of my PhD project point out an interesting correlation between VGSCs and motoneuron differentiation with possible implication in ALS pathogenesis.

VOLTAGE GATED SODIUM CHANNEL AND NEURONAL DIFFERENTIATION. IMPLICATIONS IN AMYOTROPHIC LATERAL SCLEROSIS PATHOGENESIS / M.n. Colombo ; tutor: M. Francolini ; coordinatore: N. Landsberger. - : . Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, 2022 Sep 21. ((34. ciclo, Anno Accademico 2021.

VOLTAGE GATED SODIUM CHANNEL AND NEURONAL DIFFERENTIATION. IMPLICATIONS IN AMYOTROPHIC LATERAL SCLEROSIS PATHOGENESIS

M.N. Colombo
2022

Abstract

Voltage gated sodium channels (VGSCs) are multimeric protein complexes formed by one alpha subunit, responsible of pore formation and of the regulation of the state of the channel, and by one or two accessory beta subunits that have an important role in the regulation of alpha subunit localization and kinetics and in the interaction with the extracellular environment. VGSCs, together with Calcium and Potassium voltage dependent channels, are involved in the definition of cellular electrical properties. In particular, a small percentage (1%) of the voltage dependent Na+ current generated by VGSCs is a non-inactivating component (Persistent Sodium Current, INaP). Recent evidence pointed out an involvement of VGSCs and INaP in promoting neurite elongation in health and disease. Indeed, NaV 1.6 and b1 subunits were reported to be fundamental in axonal outgrowth in mouse neuron primary culture. Moreover, our previous studies on a zebrafish model of Amyotrophic Lateral Sclerosis, that express the mutant G93R form of the superoxide dismutase 1 (SOD1) enzyme, show that alteration in the INaP of spinal inter- and moto- neurons induced neuron hyperexcitability that finally leads to hyperactive zebrafish locomotor behaviour together with morphological alterations of motoneuronal phenotype. In this scenario, we decided to deeper investigate the role of VGSCs in neurite elongation, by taking advantage of the NSC34 cell line, an in vitro model often used in the study of motoneuron disease. Thus, we used two different NSC34 model of ALS, the first one expressing the human wild-type (WT) or G93A form of the SOD1 enzyme and the second one that lacks for the expression of the vesicle-associated membrane protein associated protein B (VAPB) protein. By mean of different experimental approaches we study the expression of VGSCs protein and mRNA levels, the localization of the channel and the ability of the cell to respond to a depolarizing stimulus in health and disease in vitro models. Then, taking advantage of pharmacological approaches we evaluate whether changes in VGSCs functionality cause impairments in neurites elongation. Surprisingly, we found a strong correlation between VGSCs expression, localization and functionality and neuron differentiation in all the model we analysed. In particular, VGSCs increased expression seems to be functional to the increase in the percentage of differentiated cells during NSC34 differentiation. Moreover, NSC34 defective neurite outgrowth correlates with VGSCs defective expression or localization in NSC34 ALS models. In conclusion, the study that I have conduct during the three years of my PhD project point out an interesting correlation between VGSCs and motoneuron differentiation with possible implication in ALS pathogenesis.
FRANCOLINI, MAURA
LANDSBERGER, NICOLETTA
Settore BIO/13 - Biologia Applicata
VOLTAGE GATED SODIUM CHANNEL AND NEURONAL DIFFERENTIATION. IMPLICATIONS IN AMYOTROPHIC LATERAL SCLEROSIS PATHOGENESIS / M.n. Colombo ; tutor: M. Francolini ; coordinatore: N. Landsberger. - : . Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, 2022 Sep 21. ((34. ciclo, Anno Accademico 2021.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/937002
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