Efectos de la activación de calpaínas mediada por el receptor p2x2 sobre la vía amiloidogénica en la enfermedad de Alzheimer.
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Date
2025
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Universidad de Concepción
Abstract
La Enfermedad de Alzheimer (EA) es el principal tipo de demencia que afecta a la población adulto mayor. Se postula que el acontecimiento clave en la patogénesis de esta enfermedad es la acumulación de péptido beta-amiloide (Aβ), que produce muerte neuronal en áreas como el hipocampo, la amígdala y la corteza cerebral, causando un deterioro cognitivo severo.
Previamente, nuestro grupo demostró que tanto los aumentos en el Ca2+ intracelular, así como la disminución en los niveles proteínas sinápticas y en la viabilidad neuronal, inducidos por Aβ, eran prevenidos con un antagonista de los receptores purinérgicos P2X (P2XR). Y que, específicamente la subunidad P2X2R incrementa su expresión luego de la exposición crónica a Aβ, receptor que es altamente permeable a Ca2+. Entre las proteínas que participan en vías de señalización intracelular dependientes de Ca2+ están las calpaínas; proteasas citosólicas que pueden participar en la proteólisis de algunos sustratos para potenciar la formación de Aβ en la EA.
Dados los antecedentes, en esta tesis nos propusimos estudiar la actividad de calpaínas mediada por la activación de los receptores purinérgicos P2X2 para determinar su impacto sobre la vía amiloidogénica y formación de Aβ, en modelos de sobreexpresión de P2X2R.
Nuestros estudios en células PC-12 muestran que la activación de P2X2R produce un aumento específico en los niveles de calpaína-1 (146±6%), mientras que los niveles de calpaína-2 y el inhibidor de calpaínas endógeno: calpastatina, no se vieron afectados. La actividad proteasa de calpaínas también incrementó significativamente debido a la degradación de sus principales sustratos: espectrina (64±5%), p35 (128±3%), y el sustrato fluorescente de calpaínas Ac LLY-AFC (158±9%).
A su vez, la proteólisis de p35 produjo el fragmento p25, que activa de forma permanente a la quinasa neuronal dependiente de ciclina 5 (Cdk5). Esta quinasa aumentó los niveles de STAT3 fosforilado (363±38%), un factor de transcripción de regulación positiva para BACE1; enzima que cumple un rol clave en la vía amiloidogénica de la proteína precursora amiloide (APP). En efecto, encontramos niveles elevados de BACE1 (124±5%), y el péptido Aβ (159±4%), que se evitaron con el uso del inhibidor de calpaínas MDL-28170.
Niveles elevados de P2X2R (120±2%) y calpaína-1 (168±9%), también se encontraron en el hipocampo de ratones transgénicos APP/PS1. Sumado a la degradación de espectrina (68±5%) y p35 (165±13%), y niveles elevados de pSTAT3 (166±17%) que se asociaron con altos niveles de BACE1 (151±12%) y Aβ (132±6%), lo que implica que el eje de activación P2X2R/calpaína-1 está desregulado en este modelo animal de la EA, y probablemente contribuyendo a la acumulación de Aβ. Además, estos resultados mostraron tener una correlación con los aumentos en los niveles de P2X2R (176±22%) y calpaína-1 (150±12%) observados en muestras de corteza prefrontal de pacientes post-mortem con EA, específicamente en fracciones enriquecidas en membranas presinápticas, donde la mayor expresión de P2X2R podría contribuir a la actividad incrementada de calpaína-1 y la consecuente potenciación de la vía amiloidogénica.
En resumen, nuestros resultados nos permiten proponer al eje P2X2R/calpaína 1 como un elemento relevante en la fisiopatología de la EA. Nuestros datos apuntan a un mecanismo de retroalimentación positiva, donde el péptido Aβ incrementa la expresión de P2X2R para producir más Aβ intracelular, y este incremento estaría mediado por un mecanismo dependiente de calpaína-1. Esto podría representar un punto de partida novedoso en la elaboración de nuevas estrategias terapéuticas que contribuyan al tratamiento de la EA.
Alzheimer's disease (AD) is the main type of dementia affecting the older adult population. It is postulated that the key event in the pathogenesis of this disease is the accumulation of beta-amyloid peptide (Aβ), which produces neuronal death in areas such as the hippocampus, amygdala and cerebral cortex, causing severe cognitive impairment. Previously, our group demonstrated that increases in intracellular Ca2+, decreases in synaptic protein levels and neuronal viability induced by Aβ were prevented by a purinergic P2X receptor (P2XR) antagonist. And specifically, P2X2R subunit increases its expression after chronic exposure to Aβ, a receptor that is highly permeable to Ca2+. Among the proteins involved in intracellular Ca2+-dependent signaling pathways are calpains; cytosolic proteases that may participate in the proteolysis of some substrates to enhance Aβ formation in AD. For this reason, in this thesis we set out to study the activity of calpains mediated by P2X2 purinergic receptor activation to determine their impact on the amyloidogenic pathway and Aβ formation in P2X2R overexpression models. Our studies in PC-12 cells show that P2X2R activation produces a specific increase in calpain-1 levels (146±6%), whereas calpain-2 levels and the endogenous calpain inhibitor calpastatin were not affected. Calpain protease activity also increased significantly due to degradation of its main substrates: spectrin (64±5%), p35 (128±3%), and the fluorescent calpain substrate Ac-LLY AFC (158±9%). In turn, proteolysis of p35 produced the p25 fragment, which permanently activates neuronal cyclin-dependent kinase 5 (Cdk5). This kinase increased the levels of phosphorylated STAT3 (363±38%), a positively regulated transcription factor for BACE1; an enzyme that plays a key role in the amyloidogenic pathway of amyloid precursor protein (APP). Indeed, we found elevated levels of BACE1 (124±5%), and Aβ peptide (159±4%), which were prevented with the use of the calpain inhibitor MDL-28170. Elevated levels of P2X2R (120±2%) and calpain-1 (168±9%) were also found in the hippocampus of APP/PS1 transgenic mice. Added to the degradation of spectrin (68±5%) and p35 (165±13%), and elevated levels of pSTAT3 (166±17%) that were associated with high levels of BACE1 (151±12%) and Aβ (132±6%), implying that the P2X2R/calpain-1 activation axis is dysregulated in this animal model of AD, and probably contributing to the accumulation of Aβ. Furthermore, these results were shown to correlate with increases in P2X2R (176±22%) and calpain-1 (150±12%) levels observed in prefrontal cortex samples from postmortem AD patients, specifically in fractions enriched in presynaptic membranes, where increased P2X2R expression could contribute to increased calpain-1 activity and consequent potentiation of the amyloidogenic pathway. In summary, our results allow us to propose the P2X2R/calpain-1 axis as a relevant element in the pathophysiology of AD. Our data point to a positive feedback mechanism, where Aβ peptide increases P2X2R expression to produce more intracellular Aβ, and this increase would be mediated by a calpain-1 dependent mechanism. This could represent a novel starting point in the development of new therapeutic strategies that contribute to the treatment of AD.
Alzheimer's disease (AD) is the main type of dementia affecting the older adult population. It is postulated that the key event in the pathogenesis of this disease is the accumulation of beta-amyloid peptide (Aβ), which produces neuronal death in areas such as the hippocampus, amygdala and cerebral cortex, causing severe cognitive impairment. Previously, our group demonstrated that increases in intracellular Ca2+, decreases in synaptic protein levels and neuronal viability induced by Aβ were prevented by a purinergic P2X receptor (P2XR) antagonist. And specifically, P2X2R subunit increases its expression after chronic exposure to Aβ, a receptor that is highly permeable to Ca2+. Among the proteins involved in intracellular Ca2+-dependent signaling pathways are calpains; cytosolic proteases that may participate in the proteolysis of some substrates to enhance Aβ formation in AD. For this reason, in this thesis we set out to study the activity of calpains mediated by P2X2 purinergic receptor activation to determine their impact on the amyloidogenic pathway and Aβ formation in P2X2R overexpression models. Our studies in PC-12 cells show that P2X2R activation produces a specific increase in calpain-1 levels (146±6%), whereas calpain-2 levels and the endogenous calpain inhibitor calpastatin were not affected. Calpain protease activity also increased significantly due to degradation of its main substrates: spectrin (64±5%), p35 (128±3%), and the fluorescent calpain substrate Ac-LLY AFC (158±9%). In turn, proteolysis of p35 produced the p25 fragment, which permanently activates neuronal cyclin-dependent kinase 5 (Cdk5). This kinase increased the levels of phosphorylated STAT3 (363±38%), a positively regulated transcription factor for BACE1; an enzyme that plays a key role in the amyloidogenic pathway of amyloid precursor protein (APP). Indeed, we found elevated levels of BACE1 (124±5%), and Aβ peptide (159±4%), which were prevented with the use of the calpain inhibitor MDL-28170. Elevated levels of P2X2R (120±2%) and calpain-1 (168±9%) were also found in the hippocampus of APP/PS1 transgenic mice. Added to the degradation of spectrin (68±5%) and p35 (165±13%), and elevated levels of pSTAT3 (166±17%) that were associated with high levels of BACE1 (151±12%) and Aβ (132±6%), implying that the P2X2R/calpain-1 activation axis is dysregulated in this animal model of AD, and probably contributing to the accumulation of Aβ. Furthermore, these results were shown to correlate with increases in P2X2R (176±22%) and calpain-1 (150±12%) levels observed in prefrontal cortex samples from postmortem AD patients, specifically in fractions enriched in presynaptic membranes, where increased P2X2R expression could contribute to increased calpain-1 activity and consequent potentiation of the amyloidogenic pathway. In summary, our results allow us to propose the P2X2R/calpain-1 axis as a relevant element in the pathophysiology of AD. Our data point to a positive feedback mechanism, where Aβ peptide increases P2X2R expression to produce more intracellular Aβ, and this increase would be mediated by a calpain-1 dependent mechanism. This could represent a novel starting point in the development of new therapeutic strategies that contribute to the treatment of AD.
Description
Tesis presentada para optar al grado de doctora en Ciencias Biológicas Área Biología Celular y Molecular
Keywords
Activación enzimática, Purinas Receptores, Enfermedad de Alzheimer