El aumento de la actividad transamidante de TG2 potencia la vía amiloidogénica en modelos de la enfermedad de Alzheimer.
Loading...
Date
2023
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Universidad de Concepción
Abstract
La Enfermedad de Alzheimer (EA) es la principal forma de demencia que afecta a los adultos mayores. Se caracteriza por una muerte neuronal progresiva en zonas de hipocampo, corteza y amígdala. Se postula que uno de los principales agentes tóxicos responsables de la neurodegeneración es la agregación tóxica del oligómeros del péptido β-amiloide (AβOs), ya que inducirían los principales eventos celulares con los que cursa esta patología, tales como dishomeostasis del Ca+2, disfunción mitocondrial, y la falla sináptica. TG2 es una enzima ubicua cuya función principal es la formación de enlaces isopeptídicos entre proteínas o péptidos, cuyo sitio catalítico se activa frente a concentraciones micromolares de Ca2+. Se ha descrito un aumento de la transcripción de TG2 en diferentes modelos transgénicos de la EA, así como en corteza de pacientes con EA. En esta tesis doctoral se evaluaron cambios en los niveles de TG2 en distintos modelos de la EA, y el efecto de la función transamidante de la enzima TG2 en la agregación del péptido Aβ, y sus principales efectos tóxicos en la función mitocondrial y neuronal. Observamos que los niveles de TG2 en el hipocampo de ratones J20 de 10 meses estaban aumentados (246 ± 17%), y que la actividad transamidante estaba potenciada (159 ± 20%), en comparación con animales WT de la misma edad. Observamos un aumento significativo de la expresión de TG2 en lisado total de hipocampo (286 ± 5 %,), y en fracción mitocondrial (313 ± 4 %) de ratones APP/PS1, en comparación con los ratones WT de la misma edad. Además, neuronas del hipocampo tratadas de forma crónica con oligómeros de Aβ (AβOs, 0.5 μM) mostraron un aumento de la inmunoreactividad de TG2 (146 ± 17%) en comparación con células no tratadas. Conjuntamente, evaluamos la capacidad de TG2 para promover los procesos de agregación del péptido Aβ, comparándola con la formación “autocatalítica” previamente estandarizada por nuestro grupo de investigación. La conversión amiloide inducida por TG2, mostró seguir una cinética hiperbólica exponencial, y fue capaz de inducir agregados con un tamaño medio de 7-13 unidades (∼28, 40, 52 kDa) que presentaban una alta citotoxicidad (85 ± 3 %). En tanto que, la formación autocatalítica mostró seguir una cinética sigmoidal, formando agregados más pequeños de tamaño de medio de 7 unidades (∼28 kDa) que presentaban una menor citotoxicidad (69 ± 3 %). El tratamiento agudo con AβTG2 indujo una rápida sobrecarga de Ca2+ citosólico (300 ± 22%) y Ca2+ mitocondrial (335 ± 43%). Utilizando la técnica de PLA, observamos que la formación del complejo IP3R/VDAC1 aumentó en las neuronas expuestas a AβTG2 bajo tratamientos crónicos (220 ± 12%). Mediante registros de patch clamp, observamos un aumento en la actividad neuronal (30 min, 257 ± 14%), que disminuyó bajo tratamientos crónicos (AβTG2 24h: 30 ± 5%). Nuestros análisis in silico mostraron que el dominio catalítico de TG2 puede interactuar con el péptido Aβ en la región N-terminal (ΔG: -10.9, Kd: 9.754e-006, Energía del complejo: -1504). De forma complementaria, quisimos evaluar la expresión de TG2 frente a la inhibición parcial del complejo mitocondrial I (MCI) con el fármaco CP2 (25
mg/kg/day) en ratones APP/PS1 sintomáticos de 24 meses, tratados durante 15 meses. Observamos que CP2 fue capaz de revertir la sobrexpresión de TG2 en animales APP/PS1 en comparación a los animales no tratados. Finalmente, quisimos correlacionar estos cambios con marcadores de función mitocondrial utilizando los mismos modelos animales. Demostramos que CP2 restaura la morfología mitocondrial, y la comunicación retículo-mitocondria, utilizando reconstrucciones tridimensionales de volúmenes obtenidas por microscopía electrónica en serie. En conjunto, nuestros resultados indican que cambios en la formación de agregados de Aβ en modelos de la EA estaría asociada a un incremento de la actividad transamidante de TG2, por lo que puede ser un potencial blanco para el desarrollo de estrategias farmacológicas en el tratamiento de la EA, y respaldan adicionalmente el desarrollo de inhibidores de TG2 como una estrategia para prevenir la agregación amiloide observada en la EA. Se requerirá avanzar en estudios adicionales, que profundicen en los mecanismos que regulan la transcripción de TG2, y los elementos involucrados en la aceleración de la vía amiloidogénica durante la progresión de la EA.
Alzheimer's Disease (AD) is the primary form of dementia affecting older adults. It is characterized by progressive neuronal death in the hippocampus, cortex, and amygdala regions. It is postulated that one of the main toxic agents responsible for neurodegeneration is the toxic aggregation of β-amyloid peptide oligomers (AβOs), as they are believed to induce the major cellular events associated with this condition, such as Ca+2 dishomeostasis, mitochondrial dysfunction, and synaptic failure. TG2 is a ubiquitous enzyme whose main function is the formation of isopeptide bonds between proteins or peptides, and its catalytic site is activated in response to micromolar concentrations of Ca2+. An increase in TG2 transcription has been described in various transgenic models of AD, as well as in the cortex of AD patients. This doctoral thesis evaluated changes in TG2 levels in different AD models and the effect of TG2's transamidase function on Aβ peptide aggregation, as well as its main toxic effects on mitochondrial and neuronal function. We observed that TG2 levels in the hippocampus of 10-month-old J20 mice were increased (246 ± 17%), and transamidase activity was enhanced (159 ± 20%), compared to age-matched WT animals. There was a significant increase in TG2 expression in the total hippocampal lysate (286 ± 5%) and the mitochondrial fraction (313 ± 4%) of APP/PS1 mice, compared to age-matched WT mice. Furthermore, hippocampal neurons treated chronically with Aβ oligomers (AβOs, 0.5 μM) showed increased TG2 immunoreactivity (146 ± 17%) compared to untreated cells. We also assessed TG2's ability to promote Aβ peptide aggregation and compared it with the previously standardized "autocatalytic" formation by our research group. TG2-induced amyloid conversion followed a hyperbolic exponential kinetics and was able to induce aggregates with a mean size of 7-13 units (∼28, 40, 52 kDa) that exhibited high cytotoxicity (85 ± 3%). In contrast, autocatalytic formation followed a sigmoidal kinetics, resulting in smaller aggregates with a mean size of 7 units (∼28 kDa) and lower cytotoxicity (69 ± 3%). Acute treatment with AβTG2 induced a rapid overload of cytosolic Ca2+ (300 ± 22%) and mitochondrial Ca2+ (335 ± 43%). Using the PLA technique, we observed an increase in the IP3R/VDAC1 complex formation in neurons exposed to chronic AβTG2 treatments (220 ± 12%). Patch clamp recordings showed an increase in neuronal activity (30 min, 257 ± 14%), which decreased under chronic AβTG2 treatments (AβTG2 24h: 30 ± 5%). Our in silico analyses showed that the catalytic domain of TG2 can interact with the Aβ peptide in the N-terminal region (ΔG: -10.9, Kd: 9.754e-006, Complex Energy: -1504). Additionally, we wanted to evaluate TG2 expression in the context of partial inhibition of mitochondrial complex I (MCI) with the drug CP2 (25 mg/kg/day) in symptomatic 24-month-old APP/PS1 mice treated for 15 months. We observed that CP2 was able to reverse the overexpression of TG2 in APP/PS1 animals compared to untreated animals. Finally, we aimed to correlate these changes with mitochondrial function markers using the same animal models. We demonstrated that CP2 restores mitochondrial morphology and ER-mitochondrial communication, using three-dimensional reconstructions of volumes obtained by serial electron microscopy. In summary, our results suggest that changes in Aβ aggregation in AD models are associated with an increase in TG2 transamidase activity. This suggests that TG2 could be a potential target for pharmacological strategies in the treatment of AD and supports the development of TG2 inhibitors as a strategy to prevent amyloid aggregation observed in AD. Further studies are needed to delve into the mechanisms regulating TG2 transcription and the elements involved in accelerating the amyloidogenic pathway during AD progression.
Alzheimer's Disease (AD) is the primary form of dementia affecting older adults. It is characterized by progressive neuronal death in the hippocampus, cortex, and amygdala regions. It is postulated that one of the main toxic agents responsible for neurodegeneration is the toxic aggregation of β-amyloid peptide oligomers (AβOs), as they are believed to induce the major cellular events associated with this condition, such as Ca+2 dishomeostasis, mitochondrial dysfunction, and synaptic failure. TG2 is a ubiquitous enzyme whose main function is the formation of isopeptide bonds between proteins or peptides, and its catalytic site is activated in response to micromolar concentrations of Ca2+. An increase in TG2 transcription has been described in various transgenic models of AD, as well as in the cortex of AD patients. This doctoral thesis evaluated changes in TG2 levels in different AD models and the effect of TG2's transamidase function on Aβ peptide aggregation, as well as its main toxic effects on mitochondrial and neuronal function. We observed that TG2 levels in the hippocampus of 10-month-old J20 mice were increased (246 ± 17%), and transamidase activity was enhanced (159 ± 20%), compared to age-matched WT animals. There was a significant increase in TG2 expression in the total hippocampal lysate (286 ± 5%) and the mitochondrial fraction (313 ± 4%) of APP/PS1 mice, compared to age-matched WT mice. Furthermore, hippocampal neurons treated chronically with Aβ oligomers (AβOs, 0.5 μM) showed increased TG2 immunoreactivity (146 ± 17%) compared to untreated cells. We also assessed TG2's ability to promote Aβ peptide aggregation and compared it with the previously standardized "autocatalytic" formation by our research group. TG2-induced amyloid conversion followed a hyperbolic exponential kinetics and was able to induce aggregates with a mean size of 7-13 units (∼28, 40, 52 kDa) that exhibited high cytotoxicity (85 ± 3%). In contrast, autocatalytic formation followed a sigmoidal kinetics, resulting in smaller aggregates with a mean size of 7 units (∼28 kDa) and lower cytotoxicity (69 ± 3%). Acute treatment with AβTG2 induced a rapid overload of cytosolic Ca2+ (300 ± 22%) and mitochondrial Ca2+ (335 ± 43%). Using the PLA technique, we observed an increase in the IP3R/VDAC1 complex formation in neurons exposed to chronic AβTG2 treatments (220 ± 12%). Patch clamp recordings showed an increase in neuronal activity (30 min, 257 ± 14%), which decreased under chronic AβTG2 treatments (AβTG2 24h: 30 ± 5%). Our in silico analyses showed that the catalytic domain of TG2 can interact with the Aβ peptide in the N-terminal region (ΔG: -10.9, Kd: 9.754e-006, Complex Energy: -1504). Additionally, we wanted to evaluate TG2 expression in the context of partial inhibition of mitochondrial complex I (MCI) with the drug CP2 (25 mg/kg/day) in symptomatic 24-month-old APP/PS1 mice treated for 15 months. We observed that CP2 was able to reverse the overexpression of TG2 in APP/PS1 animals compared to untreated animals. Finally, we aimed to correlate these changes with mitochondrial function markers using the same animal models. We demonstrated that CP2 restores mitochondrial morphology and ER-mitochondrial communication, using three-dimensional reconstructions of volumes obtained by serial electron microscopy. In summary, our results suggest that changes in Aβ aggregation in AD models are associated with an increase in TG2 transamidase activity. This suggests that TG2 could be a potential target for pharmacological strategies in the treatment of AD and supports the development of TG2 inhibitors as a strategy to prevent amyloid aggregation observed in AD. Further studies are needed to delve into the mechanisms regulating TG2 transcription and the elements involved in accelerating the amyloidogenic pathway during AD progression.
Description
Tesis presentada para optar al grado de Doctor en Ciencias Biológicas Área Biología Celular y Molecular