Instituto de Biomarcadores de Patologías Moleculares (IBPM), Universidad de Extremadura, 06006 Badajoz, Spain.
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, 06006 Badajoz, Spain.
Int J Mol Sci. 2022 Oct 21;23(20):12678. doi: 10.3390/ijms232012678.
Dysregulation in calcium signaling pathways plays a major role in the initiation of Alzheimer's disease (AD) pathogenesis. Accumulative experimental evidence obtained with cellular and animal models, as well as with AD brain samples, points out the high cytotoxicity of soluble small oligomeric forms of amyloid-β peptides (Aβ) in AD. In recent works, we have proposed that Aβ-calmodulin (CaM) complexation may play a major role in neuronal Ca signaling, mediated by CaM-binding proteins (CaMBPs). STIM1, a recognized CaMBP, plays a key role in store-operated calcium entry (SOCE), and it has been shown that the SOCE function is diminished in AD, resulting in the instability of dendric spines and enhanced amyloidogenesis. In this work, we show that 2 and 5 h of incubation with 2 μM Aβ(1-42) oligomers of the immortalized mouse hippocampal cell line HT-22 leads to the internalization of 62 ± 11 nM and 135 ± 15 nM of Aβ(1-42), respectively. Internalized Aβ(1-42) oligomers colocalize with the endoplasmic reticulum (ER) and co-immunoprecipitated with STIM1, unveiling that this protein is a novel target of Aβ. Fluorescence resonance energy transfer measurements between STIM1 tagged with a green fluorescent protein (GFP) and Aβ(1-42)-HiLyte™-Fluor555 show that STIM1 can bind nanomolar concentrations of Aβ(1-42) oligomers at a site located close to the CaM-binding site in STIM1. Internalized Aβ(1-42) produced dysregulation of the SOCE in the HT-22 cells before a sustained alteration of cytosolic Ca homeostasis can be detected, and is elicited by only 2 h of incubation with 2 μM Aβ(1-42) oligomers. We conclude that Aβ(1-42)-induced SOCE dysregulation in HT-22 cells is caused by the inhibitory modulation of STIM1, and the partial activation of ER Ca-leak channels.
钙信号通路的失调在阿尔茨海默病（AD）发病机制的启动中起着重要作用。使用细胞和动物模型以及 AD 脑样本获得的累积实验证据表明，可溶性小寡聚形式的淀粉样β肽（Aβ）在 AD 中的细胞毒性很高。在最近的研究中，我们提出 Aβ-钙调蛋白（CaM）复合物可能在神经元 Ca 信号转导中起主要作用，由 CaM 结合蛋白（CaMBP）介导。STIM1 是一种公认的 CaMBP，在钙库操纵的钙内流（SOCE）中起关键作用，并且已经表明 AD 中的 SOCE 功能减弱，导致树突棘不稳定和淀粉样蛋白形成增强。在这项工作中，我们表明，用 2 μM Aβ（1-42）寡聚体孵育 HT-22 永生化小鼠海马细胞系 2 和 5 小时分别导致内化 62±11 nM 和 135±15 nM 的 Aβ（1-42）。内化的 Aβ（1-42）寡聚体与内质网（ER）共定位，并与 STIM1 共免疫沉淀，揭示该蛋白是 Aβ 的新靶标。用绿色荧光蛋白（GFP）标记的 STIM1 与 Aβ（1-42）-HiLyte™-Fluor555 之间的荧光共振能量转移测量表明，STIM1 可以在靠近 STIM1 中 CaM 结合位点的位置结合纳米摩尔浓度的 Aβ（1-42）寡聚体。在检测到细胞溶质 Ca 动态平衡的持续改变之前，HT-22 细胞中的 Aβ（1-42）内化会导致 SOCE 失调，并且仅在孵育 2 μM Aβ（1-42）寡聚体 2 小时后就会引发。我们得出结论，HT-22 细胞中 Aβ（1-42）诱导的 SOCE 失调是由 STIM1 的抑制性调节和 ER Ca 泄漏通道的部分激活引起的。
