Abstract
We have previously shown that β-amyloid (Aβ) treatment resulted in an age-dependent calpain activation leading to Tau cleavage into a neurotoxic 17-kDa fragment in a cellular model of Alzheimer disease. This detrimental cellular response was mediated by a developmentally regulated increase in membrane cholesterol levels. In this study, we assessed the molecular mechanisms by which cholesterol modulated Aβ-induced Tau cleavage in cultured hippocampal neurons. Our results indicated that these mechanisms did not involve the regulation of the binding of Aβ aggregates to the plasma membrane. On the other hand, experiments using N-methyl-d-aspartic acid receptor inhibitors suggested that these receptors played an essential role in cholesterol-mediated Aβ-dependent calpain activity and 17-kDa Tau production. Biochemical and immunocytochemical analyses demonstrated that decreasing membrane cholesterol levels in mature neurons resulted in a significant reduction of the NR1 subunit at the membrane as well as an increase in the number of large NR1, NR2A, and NR2B subunit clusters. Moreover, the majority of these larger N-methyl-d-aspartic acid receptor subunit immunoreactive spots was not juxtaposed to presynaptic sites in cholesterol-reduced neurons. These data suggested that changes at the synaptic level underlie the mechanism by which membrane cholesterol modulates developmental changes in the susceptibility of hippocampal neurons to Aβ-induced toxicity.
Highlights
Istic morphological changes in the brain, such as senile plaque formation and the assembly of intracellular neurofibrillary tangles. -Amyloid (A), the prominent molecular component of senile plaques, has long been suspected as the main initiator of the Alzheimer disease (AD) pathogenic cascade
Amount of Membrane-bound A Was Independent of Cholesterol Levels in Cultured Hippocampal Neurons—Our previous findings indicated that an age-dependent increase in membrane cholesterol modulated developmental changes in A-induced Tau cleavage by regulating Ca2ϩ influx and calpain activation [17]
To assess whether membrane cholesterol levels determine the amount of A associated with neuronal membranes, we pharmacologically reduced its levels in mature neurons to equal that of young cells and increased young neuron membrane cholesterol levels to equal that of mature ones using MBCD and a cholesterol complex, respectively, as described previously [17, 41]
Summary
Hippocampal Culture Preparation—Hippocampal neurons were isolated from embryonic day 18 Sprague-Dawley rat embryos as described previously [18]. NMDA Receptor Inhibition—NMDA receptor activity was blocked using memantine (Sigma), a universal NMDA receptor antagonist, or ifenprodil (Sigma), an NR2B-specific NMDA receptor antagonist These agents were added to the culture medium of untreated and cholesterol-modified hippocampal neurons 1 h prior to the addition of A at a final concentration of 10 and 5 M, respectively. Immunocytochemistry—Young and mature hippocampal neurons were cultured on coverslips and treated with or without cholesterol-modifying agents, after which they were fixed in 4% paraformaldehyde in PBS containing 0.12 mM sucrose for 15 min and permeabilized in 0.3% Triton X-100 in PBS for 4 min. Whole cell lysates were loaded using tubulin as an internal control, whereas membrane fractions were loaded by total protein as assessed by the modified Lowry assay [24, 25].
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