Biding of Amiylod β (1-42)-Calmodulin Complexes to Plasma Membrane Lipid Rafts in Cerebellar Granule Neurons Alters Resting Cytosolic Calcium Homeostasis

Abstract

Las balsas lipídicas son un objetivo primario en los estudios sobre la citotoxicidad del β amiloide (Aβ) en las neuronas. Los péptidos Aβ exógenos se unen a las balsas lipídicas, que a su vez desempeñan un papel clave en la captación de Aβ, lo que conduce a la formación de agregados Aβ intracelulares neurotóxicos. Por otra parte, en la enfermedad de Alzheimer (EA) se ha observado una desregulación de la homeostasis del calcio intracelular en las neuronas. En un trabajo previo, demostramos que A β(1-42), el péptido A prevalente encontrado en las placas amiloides de pacientes con EA, se une con alta afinidad a la calmodulina (CaM) purificada, con una constante de disociación 1 μM. En este trabajo, para evaluar experimentalmente la capacidad de unión de Aβ (1-42) a CaM intracelular, utilizamos cultivos primarios de neuronas granulares cerebelosas maduras (CGN) como modelo neuronal. Nuestros resultados mostraron una gran complejación de concentraciones submicromolares de dímeros de Aβ (1-42) por CaM en CGN, hasta 120 13 picomoles de Aβ (1-42) / 2,5 106 células. Utilizando imágenes de microscopía de fluorescencia, mostramos una extensa co-localización de CaM y Aβ (1-42) en balsas lipídicas en CGN teñidas con hasta 100 picomoles de monómeros de Aβ (1-42)-HiLyteTM-Fluor555. Este tratamiento redujo el calcio citosólico en reposo de la CGN madura en un medio parcialmente despolarizante de 25 μM de potasio. We conclude that the main cause of the decrease in resting cytosolic calcium is the inhibition of CGN L-type calcium channels by Aβ (1-42) dimers, whose activity is inhibited β.

Lipid rafts are a primary target in studies of β amyloid (Aβ) cytotoxicity in neurons. Exogenous A peptides β bind to lipid rafts, which in turn play a key role in A uptake, leading to the formation of neurotoxic intracellular A aggregates. On the other hand, dysregulation of intracellular calcium homeostasis in neurons has been observed in Alzheimer’s disease (AD). In a previous work, we showed that Aβ (1–42), the prevalent Aβ peptide found in the amyloid plaques of AD patients, binds with high affinity to purified calmodulin (CaM), with a dissociation constant 1 μM. In this work, to experimentally assess the Aβ (1–42) binding capacity to intracellular CaM, we used primary cultures of mature cerebellar granule neurons (CGN) as a neuronal model. Our results showed a large complexation of submicromolar concentrations of Aβ (1–42) dimers by CaM in CGN, up to 120 13 picomoles of Aβ (1–42) / 2.5 106 cells. Using fluorescence microscopy imaging, we showed an extensive co-localization of CaM and Aβ (1–42) in lipid rafts in CGN stained with up to 100 picomoles of Aβ (1–42)-HiLyteTM-Fluor555 monomers. Intracellular Aβ (1–42) concentration in this range was achieved by 2 h incubation of CGN with 2 M Aβ (1–42), and this treatment lowered the resting cytosolic calcium of mature CGN in partially depolarizing 25 μM potassium medium. We conclude that the primary cause of the resting cytosolic calcium decrease is the inhibition of L-type calcium channels of CGN by Aβ (1–42) dimers, whose activity is inhibited by CaM: Aβ (1–42) complexes bound to lipid rafts.