Evaluación de la administración de la poliamina espermidina en la supervivencia y retraso de la disfunción neuromuscular en el modelo murino de esclerosis lateral amiotrófica hSOD1G93A
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Fecha
2022
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
La esclerosis lateral amiotrófica (ELA) es una enfermedad neurodegenerativa que afecta a las
motoneuronas (MN), causando debilidad muscular progresiva seguido de la pérdida de movilidad y
la muerte de los pacientes. Hasta el día de hoy no existe una cura para esta patología, y los tratamientos
disponibles sólo son paliativos. Es por esto que encontrar tratamientos alternativos sería un cambio
importante para el mundo de la neurociencia. En nuestro laboratorio, en su búsqueda por encontrar
mecanismos o moléculas que gatillan esta enfermedad, en una publicación reciente, se demostró que
el polifosfato inorgánico (poliP) es una molécula toxica liberada por astrocitos derivados de modelos
ELA y que genera la muerte de MN. PoliP es un polianión constituido por residuos de ortofosfato
unidos por enlaces fosfoanhidro, presente en todos los organismos estudiados con un largo de cadena
de 3 hasta cientos de residuos. Debido a su neurotoxicidad a nivel extracelular es que actualmente
buscamos moléculas candidatas para interactuar con poliP y evaluar su potencial terapéutico para el
tratamiento de ELA. Es por esto, que en la presente memoria de título se usó la poliamina
espermidina, la cual está cargada positivamente por 3 grupos amino, como una alternativa para
interactuar con poliP y con el objetivo inactivar la toxicidad de poliP. La hipótesis de este trabajo es
que el tratamiento de ratones modelos de ELA hSOD1G93A (mutSOD1) con espermidina retrasa la
disfunción neuromuscular y aumenta la sobrevida debido a la reducción de los niveles tóxicos de
poliP en la medula espinal. Los ratones mutSOD1 fueron tratados con espermidina administrada en
el agua de beber desde una etapa presintomática hasta el punto final humanitario. El inicio y
progresión de los síntomas característicos de la enfermedad se evaluó mediante test de rotarod,
además de la variación de peso corporal y sobrevida. Posteriormente, se realizó una
inmunofluorescencia en tejidos de la medula espinal lumbar de ratones mutSOD1 tratados con
espermidina, en los que se cuantificó los niveles de poliP extracelular e intracelular en astrocitos y
MN, mediante el uso de una proteína recombinante denominada recPPBD y se comparó con controles
mutSOD1 sin tratamiento. Además, se realizó un ensayo in vitro para evaluar la interacción entre
poliP y espermidina usando electroforesis en geles de Urea-PAGE. Los resultados de los parámetros
para evaluar la sintomatología no mostraron diferencias significativas entre los ratones mutSOD1
tratados o no tratados con espermidina. Con respecto a los niveles de poliP en la médula espinal,
tampoco se obtuvieron diferencias. Sin embargo, no se determinó si esta poliamina llegó al sistema
nervioso central mediante la vía de administración elegida, por lo que no podemos concluir su
potencial terapéutico sobre los síntomas de la enfermedad ni la capacidad de interacción con poliP.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons (MN), causing progressive muscle weakness followed by loss of mobility and death of patients. To this day there is no cure for this pathology, and available treatments are only palliative. This is why finding alternative treatments would be a major game-changer for the world of neuroscience. In our laboratory, in its search to find mechanisms or molecules that trigger this disease, a recent publication, showed that inorganic polyphosphate (polyP) is a toxic molecule released by astrocytes derived from ALS models and that generates the death of MN. Polyp is a polyanion consisting of orthophosphate residues linked by phosphoanhydro bonds, present in all organisms studied with a chain length of 3 to hundreds of residues. Due to its neurotoxicity at the extracellular level, we are currently searching for candidate molecules to interact with polyP, and hence neutralize toxic polyP, and evaluate its therapeutic potential for the treatment of ALS. Therefore, in the present dissertation, we used polyamine spermidine, which is positively charged by 3 amino groups, as an alternative to interacting with polyP. The hypothesis of this work is that treatment of hSOD1G93A (mutSOD1) ALS model mice with spermidine delays neuromuscular dysfunction and increases survival due to the reduction of polyP levels in the spinal cord. MutSOD1 mice were treated with spermidine administered in drinking water from a presymptomatic stage to the humane endpoint. The onset and progression of symptoms characteristic of the disease was assessed by rotarod test, in addition to bodyweight variation and survival. Subsequently, immunofluorescence was performed in lumbar spinal cord tissues of mutSOD1 mice treated with spermidine, in which the levels of extracellular and intracellular polyP in astrocytes and MN were quantified using a recombinant protein called recPPBD and compared with untreated mutSOD1 controls. In addition, an in vitro assay was performed to evaluate the interaction between polyP and spermidine using Urea-PAGE gel electrophoresis. The results of the parameters to evaluate symptomatology showed no significant differences between mutSOD1 mice treated or untreated with spermidine. Concerning polyP levels in the spinal cord, no differences were obtained either. However, it was not determined whether this polyamine reached the central nervous system by the chosen route of administration, so we cannot conclude its therapeutic potential on the symptoms of the disease or the capacity of interaction with polyP.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons (MN), causing progressive muscle weakness followed by loss of mobility and death of patients. To this day there is no cure for this pathology, and available treatments are only palliative. This is why finding alternative treatments would be a major game-changer for the world of neuroscience. In our laboratory, in its search to find mechanisms or molecules that trigger this disease, a recent publication, showed that inorganic polyphosphate (polyP) is a toxic molecule released by astrocytes derived from ALS models and that generates the death of MN. Polyp is a polyanion consisting of orthophosphate residues linked by phosphoanhydro bonds, present in all organisms studied with a chain length of 3 to hundreds of residues. Due to its neurotoxicity at the extracellular level, we are currently searching for candidate molecules to interact with polyP, and hence neutralize toxic polyP, and evaluate its therapeutic potential for the treatment of ALS. Therefore, in the present dissertation, we used polyamine spermidine, which is positively charged by 3 amino groups, as an alternative to interacting with polyP. The hypothesis of this work is that treatment of hSOD1G93A (mutSOD1) ALS model mice with spermidine delays neuromuscular dysfunction and increases survival due to the reduction of polyP levels in the spinal cord. MutSOD1 mice were treated with spermidine administered in drinking water from a presymptomatic stage to the humane endpoint. The onset and progression of symptoms characteristic of the disease was assessed by rotarod test, in addition to bodyweight variation and survival. Subsequently, immunofluorescence was performed in lumbar spinal cord tissues of mutSOD1 mice treated with spermidine, in which the levels of extracellular and intracellular polyP in astrocytes and MN were quantified using a recombinant protein called recPPBD and compared with untreated mutSOD1 controls. In addition, an in vitro assay was performed to evaluate the interaction between polyP and spermidine using Urea-PAGE gel electrophoresis. The results of the parameters to evaluate symptomatology showed no significant differences between mutSOD1 mice treated or untreated with spermidine. Concerning polyP levels in the spinal cord, no differences were obtained either. However, it was not determined whether this polyamine reached the central nervous system by the chosen route of administration, so we cannot conclude its therapeutic potential on the symptoms of the disease or the capacity of interaction with polyP.
Notas
Proyecto de título (Ingeniero en Biotecnología)
Palabras clave
Esclerosis Lateral Amiotrófica, Poliamina Espermidina, Administración y Dosificación