NATIVE IN-TISSUE STRUCTURES OF AMYLOID-β PLAQUES IN A MOUSE MODEL OF ALZHEIMER’S DISEASE BY CRYOGENIC ELECTRON TOMOGRAPHY

Senile plaques composed of amyloid-β (Aβ) are a major pathological hallmark of Alzheimer’s disease (AD). Aβ fibrils prepared in-vitro or purified from post-mortem tissue have been investigated extensively revealing their atomic structure. However, the native architecture of Aβ plaques and the organisation of Aβ fibrils within the brain itself is currently unknown. Here, we developed a workflow to determine the in-tissue architecture of Aβ plaques using cryogenic correlative light and electron tomography (cryo-CLET) in the brain of a knock-in mouse model of AD (1 year old male AppNL-G-F, n=2). After injecting mice with Methoxy-X04, a fluorescent dye that labels Aβ plaques, fresh cortical tissue biopsies were vitrified by high-pressure freezing. Next, tissue cryo-sections were collected on cryo-EM grids and Methoxy-X04 stained plaques were mapped by correlative imaging to direct the collection of cryo-electron tomographic data. 3D reconstructions revealed the in-tissue molecular architecture of Aβ plaques. Aβ fibrils were organised within multiple distinct domains of varying densities, including a meshwork and parallel bundles. Numerous other macromolecular features were observed within the plaque, including liquid-like droplets. Subcellular compartments interdigitated the pathology, within and between Aβ parallel bundles. These included membranes containing higher-order protein networks, c-shaped membrane, and membrane nanopedia. The boundary of Aβ plaques was characterised by cells in close proximity to Aβ fibrils that contained an abundance of mitochondria, endoplasmic reticulum, ribosomes and microtubules. Overall, these results reveal the molecular heterogeneity of Aβ plaques and the rich diversity of cellular constituents that define the amyloid pathology of AD.