SVCT2 y sus efectos en la arborización dendrítica de motoneuronas humanas; un modelo de Esclerosis Lateral Amiotrófica (ELA) in vitro.
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Date
2024
Journal Title
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Publisher
Universidad de Concepción
Abstract
La Esclerosis Lateral Amiotrófica (ELA) es una enfermedad neurodegenerativa multifactorial caracterizada principalmente por la pérdida selectiva y progresiva de motoneuronas (MNs). Los pacientes rara vez sobreviven más de 5 años luego de la aparición de los síntomas, debido a que el conocimiento limitado de sus causas subyacentes ha dificultado el desarrollo de terapias con efectos más significativos que los modestamente desarrollados hasta ahora.
La primera mutación descubierta causante de ELA se encuentra en el gen que codifica la Superóxido Dismutasa de Cobre y Zinc 1 (SOD1), una enzima antioxidante que cataliza la dismutación del anión superóxido en oxígeno y peróxido de hidrógeno, cuyas mutaciones se convierten en la segunda causa más frecuente de la enfermedad.
En específico, la mutación SOD1G93A, el modelo más utilizado en el estudio de la enfermedad provoca en un primer momento, estrés oxidativo y reducciones en la arborización dendrítica y axonal, seguido de hiperexcitabilidad, denervación y finalmente la muerte de MNs.
En el desafío de buscar moléculas neuroprotectoras en el contexto de ELA, investigaciones anteriores han asociado a deficiencias en los niveles de vitamina C (VC), con un alto riesgo en la obtención de la enfermedad, lo que podría explicarse principalmente por sus propiedades antioxidantes, así como por el fortalecimiento que el ácido ascórbico (AA), la forma reducida de la VC y su transportador cerebral, SVCT2 provocan en la viabilidad celular y en la diferenciación y maduración de distintas células neuronales, demostrando así, sus efectos neuroprotectores.
Por lo tanto, nuestra hipótesis de trabajo se basó en que la sobreexpresión de SVCT2 promueve la recuperación de la arborización dendrítica luego del daño celular inducido por la mutación SOD1G93A en un modelo in vitro de ELA en MNs espinales humanas generadas desde células madre pluripotentes inducidas (iPSCs).
En este contexto, se demostró que la sobreexpresión de SVCT2 en neuronas enfermas, recuperó el 63,9% y el 105% de la red dendrítica y axonal, respectivamente, redes que, fueron gravemente disminuidas por la mutación SOD1G93A.
Por otra parte, en MNs la mutación causó pérdidas en la expresión de la tinción de la proteína presináptica piccolo y aumentos en la inmunofluorescencia de PSD95, probablemente fortaleciendo la excitotoxicidad. Mientras que al sobreexpresar SVCT2, las células recuperaron parcialmente los niveles de piccolo y disminuyeron la tinción de PSD95, probablemente reduciendo a su vez la excitotoxicidad.
Los efectos morfológicos y a nivel de proteínas sinápticas provocados por la mutación SOD1G93A en nuestras MNs humanas representan temporalmente a la etapa presintomática de la enfermedad.
Finalmente, la sobreexpresión de SVCT2 fortaleció la maduración morfológica de MNs sanas y recuperó la red dendrítica y axonal, previamente degenerada por la expresión de SOD1G93A en MNs humanas.
Amyotrophic Lateral Sclerosis (ALS) is a multifactorial neurodegenerative disease characterized mainly by the selective and progressive loss of motor neurons (MNs). Patients, rarely survive longer than 5 years after the onset of symptoms, because limitations in the knowledge of the underlying causes of the disease has hindered the development of therapies with more significant effects than those modestly developed so far. The first ALS-causing mutation discovered is found in the gene coding for Copper/Zinc-Superoxide Dismutase (SOD1), an antioxidant enzyme that catalyzes the dismutation of superoxide anion into oxygen and hydrogen peroxide. Mutations in the enzyme are the second most frequent cause of the disease. Specifically, the SOD1G93A mutation, the most used model for the study of the disease, initially causes oxidative stress and reductions in dendritic and axonal arborization, followed by hyperexcitability, denervation and finally, the death of MNs. In the search for neuroprotective molecules in ALS context, previous investigations have associated deficiencies in Vitamin C (VC) levels with high risk of developing ALS. This could be explained by its antioxidant properties, as by the strengthening that ascorbic acid (AA), the reduced form of VC and its brain transporter, SVCT2, cause in cell viability, and in the differentiation and maturation of different neuronal cells, thus, demonstrating their neuroprotective effects. Therefore, our working hypothesis was that SVCT2 overexpression promotes the recovery of dendritic arborization after SOD1G93A mutation-induced cell damage in an in vitro model of ALS in human spinal MNs generated from induced pluripotent stem cells (iPSCs). In this context, we show that lentiviral overexpression of SVCT2 in diseased neurons recovered 63,9% and 105% of the dendritic and axonal network, respectively, networks that were severely diminished by the mutation SOD1G93A. On the other hand, in MNs the mutation caused decreases in the expression of the presynaptic protein piccolo staining and increases in PSD95 immunofluorescence, probably strengthening excitotoxicity. While overexpressing SVCT2 in these cells partially recovered piccolo levels and decreased PSD95 staining, probably, in turn, reducing excitotoxity. The changes in morphology and at the synaptic protein level induced by the SOD1G93A mutation in our human MNs temporally represent the presymptomatic stage of the disease. Finally, SVCT2 overexpression strengthened the morphological maturation of healthy MNs and recovered the dendritic and axonal network previously degenerated by SOD1G93A expression in human MNs.
Amyotrophic Lateral Sclerosis (ALS) is a multifactorial neurodegenerative disease characterized mainly by the selective and progressive loss of motor neurons (MNs). Patients, rarely survive longer than 5 years after the onset of symptoms, because limitations in the knowledge of the underlying causes of the disease has hindered the development of therapies with more significant effects than those modestly developed so far. The first ALS-causing mutation discovered is found in the gene coding for Copper/Zinc-Superoxide Dismutase (SOD1), an antioxidant enzyme that catalyzes the dismutation of superoxide anion into oxygen and hydrogen peroxide. Mutations in the enzyme are the second most frequent cause of the disease. Specifically, the SOD1G93A mutation, the most used model for the study of the disease, initially causes oxidative stress and reductions in dendritic and axonal arborization, followed by hyperexcitability, denervation and finally, the death of MNs. In the search for neuroprotective molecules in ALS context, previous investigations have associated deficiencies in Vitamin C (VC) levels with high risk of developing ALS. This could be explained by its antioxidant properties, as by the strengthening that ascorbic acid (AA), the reduced form of VC and its brain transporter, SVCT2, cause in cell viability, and in the differentiation and maturation of different neuronal cells, thus, demonstrating their neuroprotective effects. Therefore, our working hypothesis was that SVCT2 overexpression promotes the recovery of dendritic arborization after SOD1G93A mutation-induced cell damage in an in vitro model of ALS in human spinal MNs generated from induced pluripotent stem cells (iPSCs). In this context, we show that lentiviral overexpression of SVCT2 in diseased neurons recovered 63,9% and 105% of the dendritic and axonal network, respectively, networks that were severely diminished by the mutation SOD1G93A. On the other hand, in MNs the mutation caused decreases in the expression of the presynaptic protein piccolo staining and increases in PSD95 immunofluorescence, probably strengthening excitotoxicity. While overexpressing SVCT2 in these cells partially recovered piccolo levels and decreased PSD95 staining, probably, in turn, reducing excitotoxity. The changes in morphology and at the synaptic protein level induced by the SOD1G93A mutation in our human MNs temporally represent the presymptomatic stage of the disease. Finally, SVCT2 overexpression strengthened the morphological maturation of healthy MNs and recovered the dendritic and axonal network previously degenerated by SOD1G93A expression in human MNs.
Description
Tesis presentada para optar al grado de Magíster en Neurobiología
Keywords
Neurobiología, Esclerosis lateral amiotrófica, Motoneuronas