Kumar Sathish, Kapadia Akshay, Theil Sandra, Joshi Pranav, Riffel Florian, Heneka Michael T, Walter Jochen
Department of Neurology, University of Bonn Medical Center, Bonn, Germany.
Department of Neurodegenerative Diseases and Geropsychiatry, Neurology, University of Bonn Medical Center, Bonn, Germany.
Front Mol Neurosci. 2021 Jan 15;13:619639. doi: 10.3389/fnmol.2020.619639. eCollection 2020.
Aggregation and deposition of amyloid-β (Aβ) peptides in extracellular plaques and in the cerebral vasculature are prominent neuropathological features of Alzheimer's disease (AD) and closely associated with the pathogenesis of AD. Amyloid plaques in the brains of most AD patients and transgenic mouse models exhibit heterogeneity in the composition of Aβ deposits, due to the occurrence of elongated, truncated, and post-translationally modified Aβ peptides. Importantly, changes in the deposition of these different Aβ variants are associated with the clinical disease progression and considered to mark sequential phases of plaque and cerebral amyloid angiopathy (CAA) maturation at distinct stages of AD. We recently showed that Aβ phosphorylated at serine residue 26 (pSer26Aβ) has peculiar characteristics in aggregation, deposition, and neurotoxicity. In the current study, we developed and thoroughly validated novel monoclonal and polyclonal antibodies that recognize Aβ depending on the phosphorylation-state of Ser26. Our results demonstrate that selected phosphorylation state-specific antibodies were able to recognize Ser26 phosphorylated and non-phosphorylated Aβ with high specificity in enzyme-linked immunosorbent assay (ELISA) and Western Blotting (WB) assays. Furthermore, immunofluorescence analyses with these antibodies demonstrated the occurrence of pSer26Aβ in transgenic mouse brains that show differential deposition as compared to non-phosphorylated Aβ (npAβ) or other modified Aβ species. Notably, pSer26Aβ species were faintly detected in extracellular Aβ plaques but most prominently found intraneuronally and in cerebral blood vessels. In conclusion, we developed new antibodies to specifically differentiate Aβ peptides depending on the phosphorylation state of Ser26, which are applicable in ELISA, WB, and immunofluorescence staining of mouse brain tissues. These site- and phosphorylation state-specific Aβ antibodies represent novel tools to examine phosphorylated Aβ species to further understand and dissect the complexity in the age-related and spatio-temporal deposition of different Aβ variants in transgenic mouse models and human AD brains.
淀粉样β（Aβ）肽在细胞外斑块和脑血管中的聚集与沉积是阿尔茨海默病（AD）的突出神经病理学特征，且与AD的发病机制密切相关。由于存在延长、截短和翻译后修饰的Aβ肽，大多数AD患者和转基因小鼠模型大脑中的淀粉样斑块在Aβ沉积物组成上表现出异质性。重要的是，这些不同Aβ变体沉积的变化与临床疾病进展相关，并被认为标志着AD不同阶段斑块和脑淀粉样血管病（CAA）成熟的连续阶段。我们最近发现，丝氨酸残基26处磷酸化的Aβ（pSer26Aβ）在聚集、沉积和神经毒性方面具有独特特征。在本研究中，我们开发并全面验证了根据Ser26磷酸化状态识别Aβ的新型单克隆和多克隆抗体。我们的结果表明，所选的磷酸化状态特异性抗体能够在酶联免疫吸附测定（ELISA）和蛋白质印迹（WB）分析中以高特异性识别Ser26磷酸化和未磷酸化的Aβ。此外，用这些抗体进行的免疫荧光分析表明，在转基因小鼠大脑中存在pSer26Aβ，与未磷酸化的Aβ（npAβ）或其他修饰的Aβ种类相比，其沉积存在差异。值得注意的是，在细胞外Aβ斑块中微弱检测到pSer26Aβ种类，但最显著地在神经元内和脑血管中发现。总之，我们开发了新的抗体，可根据Ser26的磷酸化状态特异性区分Aβ肽，这些抗体适用于ELISA、WB以及小鼠脑组织的免疫荧光染色。这些位点和磷酸化状态特异性的Aβ抗体代表了新的工具，用于检测磷酸化的Aβ种类，以进一步理解和剖析转基因小鼠模型和人类AD大脑中不同Aβ变体在年龄相关和时空沉积方面的复杂性。
