Visualization of neurofibrillary tangle maturity in Alzheimer's disease: A clinicopathologic perspective for biomarker research

Abstract

Neurofibrillary tangles, one of the neuropathologic hallmarks of Alzheimer's disease, have a dynamic lifespan of maturity that associates with progressive neuronal dysfunction and cognitive deficits. As neurofibrillary tangles mature, the biology of the neuron undergoes extensive changes that may impact biomarker recognition and therapeutic targeting. Neurofibrillary tangle maturity encompasses three levels: pretangles, mature tangles, and ghost tangles. In this review, we detail distinct and overlapping characteristics observed in the human brain regarding morphologic changes the neuron undergoes, conversion from intracellular to extracellular space, tau immunostaining patterns, and tau isoform expression changes across the lifespan of the neurofibrillary tangle. Post-translational modifications of tau such as phosphorylation, ubiquitination, conformational events, and truncations are discussed to contextualize tau immunostaining patterns. We summarize accumulated and emerging knowledge of neurofibrillary tangle maturity, discuss the current tools used to interpret the dynamic nature in the postmortem brain, and consider implications for cognitive dysfunction and tau biomarkers.

Keywords: Alzheimer's disease; neurofibrillary tangles; neuropathology; tau; tau positron emission tomography.

FIGURE 1

Historical overview of special stains, antibody development, biomarkers, and Alzheimer's disease (AD)/neurofibrillary tangle maturity research milestones from 1875 to 2020. Blue, staining technique developed; purple, milestone research for AD; orange, antibody development; green, biomarker development. Tick marks denote 1‐year interval.

FIGURE 2

Tau structure mapped with tau antibody epitopes. Diagonal stripe denotes alternatively spliced region. Within each subheading the named tau antibody is placed corresponding to its recognition motif with associated references.

FIGURE 3

Neurofibrillary tangle (NFT) maturity level and neuritic morphologies in the CA1 subsector of the hippocampus. Pretangles have perinuclear accumulation of tau, in addition to a diffuse or granular staining pattern. Mature tangles assume the shape of the neuron in which they reside (e.g., “flame‐shaped”) and are composed of tightly packed bundles of fibers. The neuron has a dislocated and/or shrunken nucleus. Ghost tangles are loosely arranged bundles of fibers with no associated nucleus as they are the remnants of mature tangles once the neuron has died. Neuropil threads are the thread‐like staining pattern outside of the soma. Neuritic plaques are the accumulation of dystrophic neurites and are larger than a neuron. Tangle associated neuritic clusters (TANCs) are the accumulation of neurites around a ghost tangle and are around the same size as a neuron. AT8 was used for pretangle, neuropil threads, and TANCs. PHF‐1 was used for mature tangles and neuritic plaques. Ab39 was used for ghost tangles. It is important to note that these antibodies recognize a range of the NFT maturity levels. Immunohistochemistry was counterstained with hematoxylin. Arrows point to the labeled pathology. Bracket includes the entire neuritic plaque structure. Scale bar measures 25 μm.

FIGURE 4

Special stains and tau immunohistochemistry for neurofibrillary tangle (NFT) maturity levels. Representative images for each staining technique were taken at the CA1 sector of the hippocampus in Alzheimer's disease (AD) brains. Routine and special stains include Bielschowsky silver stain, H&E, Congo red, thioflavin‐S (Thio‐S), and thiazin red. All routine and special stains recognized mature tangles and ghost tangles. For immunohistochemistry, Alz‐50, MC‐1, CP13, AT8, PHF‐1, pS396, TauC3, GT‐38, Ab39, and MN423 were used. Tau antibodies recognized a range of NFT maturity levels. Immunohistochemistry was counterstained with hematoxylin. We thank Dr. García‐Sierra for the thiazin red image. Arrow head, pretangles; closed arrow, mature tangles; open arrow, ghost tangles. Scale bar measures 25 μm.

FIGURE 5

Summary of neurofibrillary tangle (NFT) maturity levels from early to advanced forms. Intermediaries exist between the defined NFT maturity levels. NFTs in intermediary 1 will have focal accumulation of fibrillar tau that does not completely fill the neuron, which was previously speculated to be nucleation sites. Intermediary 2 will have intense staining of the NFT without a nucleus. There is a shift in NFT localization, as pretangles and mature tangles are intracellular and ghost tangles are extracellular. Additionally, there is a shift in isoform expression, as pretangles predominantly express 4R tau and ghost tangles predominantly express 3R tau. Staining methods will label a range of NFT maturity levels, as identified by the dark blue triangles. Thioflavin‐S (Thio‐S) and thiazin red recognize primarily mature tangle sand ghost tangles. CP13, AT8, Alz‐50, and MC‐1 primarily recognize primarily earlier NFT maturity levels. PHF‐1, pS396, and TauC3 primarily recognize a middling NFT maturity level. Finally, Ab39, GT‐38, and MN423 primarily recognize advanced NFT maturity levels. Scale bar measures 25 μm.

FIGURE 6

Proposed tau conformational and truncation events hypothesized along the neurofibrillary tangle (NFT) lifespan. First, the N‐terminus binds to the microtubule binding region (MTBR), as recognized by Alz‐50. Next, the C‐terminus is truncated at D421, as recognized by Tau‐C3. Next, the N‐terminus is truncated, and the proline‐rich region will bind to the microtubule binding region, as recognized by TauC3. Then, tau is truncated at E391, as recognized by MN423. Finally, the proline‐rich region is further cleaved. These conformational and truncation events occur through the NFT lifespan. For example, pretangles are predominantly Alz‐50 positive, but not MN423 positive. Mature tangles are positive for Alz‐50, TauC3, Tau‐66, and MN423. Ghost tangles are predominantly positive for TauC3, Tau‐66, and MN423. Sequence of events figure reprinted from Biochim Biophys Acta, 1739, Binder et al., Tau, tangles, and Alzheimer's disease, 216‐223, Copyright (2005), with permission from Elsevier.

FIGURE 7

Tau positron emission tomography (PET) imaging of Alzheimer's type tau pathology and corresponding hippocampal tau pathology. Coronal and axial flortaucipir PET scans shown in the top panel were overlaid onto structural magnetic resonance imaging of two study participants who later came to autopsy. The left panel from a Braak stage III study participant diagnosed with diffuse Lewy body disease shows minimal flortaucipir uptake (below threshold) throughout the brain with slightly more prominence in white matter that is likely nonspecific. The corresponding histology image from PHF‐1 immunostained hippocampus revealed scattered pretangles, mature tangles, and rare ghost tangles. In contrast, the Braak VI study participant diagnosed with AD in the right panel demonstrated striking flortaucipir uptake with more intense tau PET signal (warmer red and orange color overlay) throughout the brain. The corresponding PHF‐1 immunostained hippocampus and parietal cortex reveals frequent mature tangles and ghost tangles compared to the Braak III patient. Neuritic pathology including neuropil threads and neuritic plaques is also readily observed in areas of tangle pathology. As the neuropathologic underpinnings of tau PET signal remains an intense area of investigation, the comparative contribution of tangles and neuritic pathology is still unknown. Immunohistochemistry was counterstained with hematoxylin. Black closed arrow, mature tangles; black open arrow, ghost tangles; white arrow head, neuropil thread; white closed arrow, neuritic plaque. Scale bar measures 50 μm.