An investigation of pathological tau aggregates in the postmortem human brain tissue of Alzheimer's disease patients using correlative light and electron microscopy

Alzheimer's disease (AD) is a progressive, neurodegenerative disease and the most common form of dementia. Despite more than a hundred years of research, there is no cure for AD. The pathological aggregation of the protein tau to form intraneuronal neurofibrillary tangles (NFTs) and of Aß into extracellular plaques is a major hallmark of AD. NFTs have been classified into pretangles, mature tangles, and ghost tangles based on staining profiles for different amyloid dyes and tau markers detected by immunohistochemistry, which, however, provides no ultrastructural information. Electron microscopy (EM) has long been the gold standard for visualizing ultrastructure and was instrumental in the initial identification of tau paired helical filaments in AD. Correlative light and electron microscopy (CLEM) bridges this gap by preserving both molecular specificity, via immunolabeling, and ultrastructural detail, via EM, in the same specimen. In Chapter 1, I present a room-temperature CLEM workflow enabling precise mapping of disease-relevant protein aggregates within post-mortem human brain, setting the stage for the ultrastructural studies that follow in Chapters 2 and 3. In Chapter 2, I apply this CLEM workflow to characterize the ultrastructure of different levels of NFT maturation. Mature tangles contained highly aligned, clustered twisting tau fibrils that were mostly devoid of membranous material, while pretangles showed no ultrastructural differences to control cells in the same tissue. Multilamellar bodies could be occasionally found in mature tangles but to a much lesser degree than in dystrophic neurites. Dystrophic neurites were swollen and showed a continuum of pathology, which was either predominantly membranous or fibrillar. Ghost tangles were highly compartmentalized, most likely due to astrocytic infiltration. Fibrils were highly aligned and densely packed. Interestingly, they lacked a twist and were thinner than fibrils in mature tangles. Based on the staining profile and the morphology of these ghost tangle fibrils, it seems likely tau fibrils are cleaved and the two protofilament subunits fall apart as NFTs progress from mature tangle to ghost tangle. More recently, the atomic structures of patient-derived tau fibrils have been solved. However, due to the extraction of fibrils from tissue, all spatial context is lost in these studies. Cryo-CLEM coupled with cryo-electron tomography allows for sub-nanometer studies in situ. Chapter 3 presents preliminary results of a cryo-CLEM pipeline adapted to chemically-fixed, post-mortem AD brain. This pipeline allows the visualization of tau fibrils. This opens the door to future studies that link molecular epitope identity, ultrastructural morphology, and atomic-level structure in post-mortem human AD tissue. Together, this thesis provides an adapted technical platform for correlative light and electron microscopy and applies it directly to a central biological question in Alzheimer's disease: By