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

The proteome of granulovacuolar degeneration and neurofibrillary tangles in Alzheimer’s disease

Granulovacuolar degeneration (GVD) is a common feature in Alzheimer’s disease (AD). The occurrence of GVD is closely associated with that of neurofibrillary tangles (NFTs) and GVD is even considered to be a pre-NFT stage in the disease process of AD. Currently, the composition of GVD bodies, the mechanisms associated with GVD and how GVD exactly relates to NFTs is not well understood. By combining immunohistochemistry (IHC) and laser microdissection (LMD) we isolated neurons with GVD and those bearing tangles separately from human post-mortem AD hippocampus (n = 12) using their typical markers casein kinase (CK)1δ and phosphorylated tau (AT8). Control neurons were isolated from cognitively healthy cases (n = 12). 3000 neurons per sample were used for proteome analysis by label free LC–MS/MS. In total 2596 proteins were quantified across samples and a significant change in abundance of 115 proteins in GVD and 197 in tangle bearing neurons was observed compared to control neurons. With IHC the presence of PPIA, TOMM34, HSP70, CHMP1A, TPPP and VXN was confirmed in GVD containing neurons. We found multiple proteins localizing specifically to the GVD bodies, with VXN and TOMM34 being the most prominent new protein markers for GVD bodies. In general, protein groups related to protein folding, proteasomal function, the endolysosomal pathway, microtubule and cytoskeletal related function, RNA processing and glycolysis were found to be changed in GVD neurons. In addition to these protein groups, tangle bearing neurons show a decrease in ribosomal proteins, as well as in various proteins related to protein folding. This study, for the first time, provides a comprehensive human based quantitative assessment of protein abundances in GVD and tangle bearing neurons. In line with previous functional data showing that tau pathology induces GVD, our data support the model that GVD is part of a pre-NFT stage representing a phase in which proteostasis and cellular homeostasis is disrupted. Elucidating the molecular mechanisms and cellular processes affected in GVD and its relation to the presence of tau pathology is highly relevant for the identification of new drug targets for therapy