Calcium sensing receptor mediated the excessive generation of β-amyloid peptide induced by hypoxia in vivo and in vitro

Highlights

• Hypoxia up-regulated calcium sensing receptor (CaSR) expression in hippocampal neurons and tissue.

• Hypoxia elevated [Ca²⁺]_i through calcium sensing receptor to promote the expression of BACE1.

• Calcium sensing receptor mediated Aβ overproduction induced by hypoxia.

Abstract

Hypoxia played an important role in the pathogenesis of AD. Hypoxia increased Aβ formation, then caused Alzheimer's disease. Calcium sensing receptor (CaSR) was involved in the regulation of cell growth, differentiation, hormonal secretion and other physiological function. Increasing evidence supported CaSR might play a more prominent role in susceptibility to AD, but the role of CaSR in Aβ overproduction induced by hypoxia and its mechanisms remain unclear. To investigate whether CaSR mediated the overproduction of Aβ induced by hypoxia, immunoblot and immunochemistry were employed to determine the expression of CaSR and BACE1 in hippocampal neurons and tissue and Ca²⁺ image system was used to measure [Ca²⁺]_i in hippocampal neurons. The content of Aβ was detected with ELISA kits. Our research found that hypoxia increased the expression of CaSR in hippocampal neurons and tissue and [Ca²⁺]_i in hippocampal neurons. Calhex 231, a selective blocher of CaSR, inhibited the increase in [Ca²⁺]_i induced by hypoxia. Hypoxia or GdCl₃, an agonist of CaSR, increased the expression of BACE1 in hippocampal neurons and tissue, but Calhex 231 or Xesto C (a selective inhibitor of IP₃ receptor) partly prevented hypoxia-induced BACE1 overexpression. Hypoxia or GdCl₃ increased the content of Aβ₄₂ and Aβ₄₀ in hippocampal tissue, however Calhex 231 or Xesto C prevented hypoxia-induced the overproduction of Aβ₄₂ and Aβ₄₀ partly. Based on the above data, we suggested that hypoxia increased [Ca²⁺]_i by elevated CaSR expression to promote BACE1 expression, thereby resulting in the overproduction of Aβ₄₂ and Aβ₄₀.

Introduction

With the life expectancy increasing, Alzheimer's disease (AD) shows a trend of sharp rise in the incidence of its attack. The pathogenesis of AD is still unclear and there is not effective measure to treat AD, so it is particularly important to study the pathogenesis of AD. Senile plaque (SP), one of AD pathology feature, was formed by the deposition of β-amyloid peptide (Aβ) [1]. Amyloid precursor protein (APP) is cleaved by β-secretase (β site APP cleaving enzyme, BACE) and γ-secretase to generate Aβ [2]. A rise in the APP and/or BACE and γ-secretase activity increases the formation of Aβ.

Hypoxia is a frequent pathological process and plays an important role in the pathogenesis of AD. Studies showed that hypoxic injury, such as cerebral ischemia and stroke, significantly increased in the incidence of AD [3], [4], [5]. Hypoxia caused the increase of cytoplasm calcium concentration ([Ca²⁺]_i) [6], [7], and elevated [Ca²⁺]_i promoted the formation of Aβ [8]. However, the mechanisms of Aβ overproduction induced by hypoxia are still unclear.

Calcium sensing receptor (CaSR) was firstly separated and cloned from bovine parathyroid gland [9], and identified as the G protein coupled receptor. Brown et al. reported the activation of CaSR triggered a rise of [Ca²⁺]_i and related signal transduction pathways to inhibit the secretion of parathyroid hormone, therefor feeding back to regulate extracellular calcium concentration when extracellular calcium concentration was increased [9]. CaSR was expressed not only in parathyroid tissue but also in neural tissue [10], [11] and its other functions, such as regulation of cell growth, differentiation, apoptosis, gene expression and cell secretion, were continuously found [12], [13], [14]. Studies demonstrated that CaSR might play a more prominent role in susceptibility to AD among individuals that lacked an APOE4 allele [15], but it is still unclear about the role and mechanisms of CaSR in Aβ overproduction induced by hypoxia.

The present study explored the effect of hypoxia on CaSR expression and the role of CaSR in the change of [Ca²⁺]_i and BACE1 expression induced by hypoxia, and researched the effects of the change of CaSR expression and [Ca²⁺]_i on hypoxia-induced Aβ overproduction.