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

Joana Poejo,Ana M Mata,Jairo Salazar,Carlos Gutierrez-Merino

doi:10.3390/ijms22041984

Abstract

Lipid rafts are a primary target in studies of amyloid β (Aβ) cytotoxicity in neurons. Exogenous Aβ peptides bind to lipid rafts, which in turn play a key role in Aβ uptake, leading to the formation of neurotoxic intracellular Aβ 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 nM. 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 μM Aβ(1–42), and this treatment lowered the resting cytosolic calcium of mature CGN in partially depolarizing 25 mM 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.

Highlights

Exogenous Amyloid β (Aβ) peptides bind to lipid rafts, which in turn play a key role in Aβ uptake, leading to the formation of neurotoxic intracellular Aβ aggregates
Aβ(1–42) is the prevalent Aβ peptide found in the amyloid plaques of Alzheimer’s disease (AD) patients [12], and it has been shown that reuptake of extracellular Aβ(1–42) into neurons can lead to the formation of intracellular aggregates, resulting in published maps and institutional affiliations
Since Aβ(1–42) has been shown to bind to lipid rafts, before running the co-immunoprecipitation assays, the cerebellar granule neurons (CGN) lysates were treated with 10 mM methyl-β-cyclodextrin during 30 min to solubilize lipid rafts and allow for dissociation of the proteins associated with these subcellular structures

Summary

Introduction

Exogenous Aβ peptides bind to lipid rafts, which in turn play a key role in Aβ uptake, leading to the formation of neurotoxic intracellular Aβ aggregates. Aβ(1–42) is the prevalent Aβ peptide found in the amyloid plaques of AD patients [12], and it has been shown that reuptake of extracellular Aβ(1–42) into neurons can lead to the formation of intracellular aggregates, resulting in published maps and institutional affiliations. It is to be remarked that oligomeric species of Aβ(1–42) are tightly linked to AD pathogenesis and are presumed to be the cause of neuronal damage [16] This hypothesis is further supported by the results obtained with the triple transgenic (3xTg) AD mice, where the level of intraneuronal Aβ correlates with synaptic dysfunction and memory impairment [17,18]

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