Department of Neurosurgery, University Hospital Bonn, Bonn, Germany.
Department of Pharmaceutics, Institute of Pharmacy, University of Bonn, Bonn, Germany.
Fluids Barriers CNS. 2023 Nov 22;20(1):85. doi: 10.1186/s12987-023-00492-7.
Parenchymal accumulation of beta-amyloid (Aβ) characterizes Alzheimer's disease (AD). Aβ homeostasis is maintained by two ATP-binding cassette (ABC) transporters (ABCC1 and ABCB1) mediating efflux, and the receptor for advanced glycation end products (RAGE) mediating influx across the blood-brain barrier (BBB). Altered transporter levels and disruption of tight junctions (TJ) were linked to AD. However, Aβ transport and the activity of ABCC1, ABCB1 and RAGE as well as the functionality of TJ in AD are unclear.
ISMICAP, a BBB model involving microperfusion of capillaries, was used to assess BBB properties in acute cortical brain slices from Tg2576 mice compared to wild-type (WT) controls using two-photon microscopy. TJ integrity was tested by vascularly perfusing biocytin-tetramethylrhodamine (TMR) and quantifying its extravascular diffusion as well as the diffusion of FM1-43 from luminal to abluminal membranes of endothelial cells (ECs). To assess ABCC1 and ABCB1 activity, calcein-AM was perfused, which is converted to fluorescent calcein in ECs and gets actively extruded by both transporters. To probe which transporter is involved, probenecid or Elacridar were applied, individually or combined, to block ABCC1 and ABCB1, respectively. To assess RAGE activity, the binding of 5-FAM-tagged Aβ by ECs was quantified with or without applying FPS-ZM1, a RAGE antagonist.
In Tg2576 mouse brain, extravascular TMR was 1.8-fold that in WT mice, indicating increased paracellular leakage. FM1-43 staining of abluminal membranes in Tg2576 capillaries was 1.7-fold that in WT mice, indicating reduced TJ integrity in AD. While calcein was undetectable in WT mice, its accumulation was significant in Tg2576 mice, suggesting lower calcein extrusion in AD. Incubation with probenecid or Elacridar in WT mice resulted in a marked calcein accumulation, yet probenecid alone had no effect in Tg2576 mice, implying the absence of probenecid-sensitive ABC transporters. In WT mice, Aβ accumulated along the luminal membranes, which was undetectable after applying FPS-ZM1. In contrast, marginal Aβ fluorescence was observed in Tg2576 vessels, and FPS-ZM1 was without effect, suggesting reduced RAGE binding activity.
Disrupted TJ integrity, reduced ABCC1 functionality and decreased RAGE binding were identified as BBB alterations in Tg2576 mice, with the latter finding challenging the current concepts. Our results suggest to manage AD by including modulation of TJ proteins and Aβ-RAGE binding.
β-淀粉样蛋白（Aβ）在大脑中的积累是阿尔茨海默病（AD）的特征。Aβ 的体内平衡由两种 ATP 结合盒（ABC）转运蛋白（ABCC1 和 ABCB1）介导外排，以及晚期糖基化终产物受体（RAGE）介导通过血脑屏障（BBB）的内流来维持。转运蛋白水平的改变和紧密连接（TJ）的破坏与 AD 有关。然而，Aβ 转运以及 ABCC1、ABCB1 和 RAGE 的活性以及 AD 中 TJ 的功能尚不清楚。
使用双光子显微镜，通过微灌注毛细血管，在急性皮质脑切片中评估 Tg2576 小鼠与野生型（WT）对照之间的 BBB 特性。通过血管内灌注生物胞嘧啶四甲基罗丹明（TMR）并量化其血管外扩散以及 FM1-43 从内皮细胞（ECs）的腔膜到基底外侧膜的扩散来测试 TJ 完整性。为了评估 ABCC1 和 ABCB1 的活性，用 calcein-AM 进行灌注，该物质在 ECs 中转化为荧光 calcein，并被这两种转运蛋白主动外排。为了探究哪种转运蛋白参与其中，分别或联合应用丙磺舒或 Elacridar 来阻断 ABCC1 和 ABCB1。为了评估 RAGE 活性，用 ECs 结合的 5-FAM 标记的 Aβ 进行定量，无论是否应用 RAGE 拮抗剂 FPS-ZM1。
在 Tg2576 小鼠的大脑中，TMR 的血管外扩散是 WT 小鼠的 1.8 倍，表明细胞旁渗漏增加。Tg2576 毛细血管基底外侧膜的 FM1-43 染色是 WT 小鼠的 1.7 倍，表明 AD 中 TJ 完整性降低。虽然在 WT 小鼠中未检测到 calcein，但在 Tg2576 小鼠中 calcein 明显积累，表明 AD 中 calcein 外排减少。在 WT 小鼠中，丙磺舒或 Elacridar 的孵育导致 calcein 明显积累，但丙磺舒单独在 Tg2576 小鼠中没有效果，暗示缺乏丙磺舒敏感的 ABC 转运蛋白。在 WT 小鼠中，Aβ 积累在腔膜上，而应用 FPS-ZM1 后则无法检测到。相比之下，在 Tg2576 血管中仅观察到边缘 Aβ 荧光，而 FPS-ZM1 没有效果，表明 RAGE 结合活性降低。
在 Tg2576 小鼠中发现 TJ 完整性受损、ABCC1 功能降低和 RAGE 结合减少，这是 BBB 改变的特征，后一种发现对当前的概念提出了挑战。我们的研究结果表明，通过调节 TJ 蛋白和 Aβ-RAGE 结合，可以治疗 AD。
