The amyloid β-protein (Aβ) is believed to play an initiating role in Alzheimer’s disease (AD) and aqueous extracts of certain AD brains can cause Aβ-dependent toxicity. When examined using denaturing SDS-PAGE such extracts contain two broad bands at ~4 and ~7kDa. While ~4kDa-Aβ has been shown to contain a variety of Aβ primary structures, the identity of ~7kDa-Aβ, has been controversial. Here, we report methods that allowed the identification of components of ~7kDa Aβ.
~4kDa-Aβ and ~7kDa-Aβ were isolated from the aqueous phase of human brain and solubilized amyloid plaques. Long-term potentiation (LTP) and live-cell imaging of iPSC-derived neurons was used to assess bioactivity, and portions of the same materials were used for mass spectrometry. Analysis of intact and proteolytically digested ~4kDa-Aβ and ~7kDa-Aβ was performed using LC-MS/MS under both acidic and alkaline conditions. Several types of database search softwares, in-house written scripts, and manual analysis, were utilized to identify ~7kDa-Aβ.
~7kDa-Aβ, but not ~4kDa-Aβ, blocked hippocampal LTP and disrupted the neuritic architecture of iPSC-derived neurons. Analysis of ~4kDa-Aβ from six separate AD brains identified more than 35 Aβ primary structures - including the previously elusive p3 fragment, Aβ17-42. When alkaline conditions were used to analysis ~7kDa-Aβ solubilized from plaques, 11 covalently cross-linked Aβ heterodimers were detected. These included: 1-38×1-40, 1-40×1-40, 2-40×1-40, and 1-42×1-42. Intriguingly, LC-MS/MS analysis of trypsin-treated ~7kDa-Aβ identified Aβ dimers linked between Asp1 and Glu22.
Our demonstration of covalently cross-linked Aβ heterodimers in human brain recommends further investigation of dimers as therapeutic targets and potential AD biomarkers. However, large amounts of material were required for our analysis and only the most abundant dimers were positively identified. In future studies it will be essential to increase analytical sensitivity and develop software to allow the identification of low abundant dimers.