Super-Resolution Imaging of Alzheimer's Disease Hyperphosphorylated Tau Aggregates

项目总结

Project Summary Alzheimer’s disease (AD) is a neurological disorder characterized by the accumulation of microtubule- associated protein tau into many abnormal intraneuronal aggregates detected at distinct brain regions. Their emergence strongly correlates with the progressive severity of AD diagnosed patients’ clinical symptoms and neuropathological features. These observations suggest tau aggregation plays a crucial and toxic role in AD. Tau is a monomeric highly soluble protein that maintains the assembly and stability of microtubules. Tau’s function is regulated through post-translational modifications (PTMs) of a specific number of residues, primarily phosphorylation. Previous studies indicate that the tau proteins that make up polymorphous insoluble tau aggregates (e.g., neurofibrillary tangles) have abnormal hyperphosphorylation. Based on these observations, it is suggested that ubiquitous hyperphosphorylation of tau promotes its aggregation in disease. However, the precise link between the pattern and degree of tau hyperphosphorylation with aggregation is unclear. Furthermore, how a heavily molecularly modified protein can form a wide range of morphologically diverse aggregates within one disease is not well-understood. These questions have not been investigated thoroughly due to the diffraction limit of conventional light microscopy (~250nm), in which aggregates of a size well below this limit are not resolvable. By labeling human postmortem AD brain tissues with multiple hyperphosphorylation- specific phosphor-tau antibodies and super-resolution imaging, I have been able to identify tau oligomers ( 20- 30 nm), linear fibrils (30-250 nm), branched fibrils (50-350 nm), and NFTs (>1μm). Based on this preliminary data, I hypothesize that the tau oligomers/small fibrils present in AD have unique PTM profiles that arise from the combination of tau proteins hyperphosphorylated at different residues. I also hypothesize that morphologically distinct insoluble tau aggregates in AD have unique PTM profiles and that these profiles match the PTM profiles of tau oligomers/small fibrils. To test these hypotheses, the aims established in this proposal will determine the relative frequency of tau proteins carrying a particular array of hyperphosphorylated sites within tau oligomers, fibrils, and NFTs. I will achieve this by combinatorial immunostaining with established phosphor-tau antibodies, multicolor super-resolution imaging, and advanced quantitative analysis using machine learning to rigorously evaluate the hyperphosphorylation profiles of all tau aggregates present in human AD tissues. These studies will demonstrate that it is possible to determine the PTM profiles of tau oligomers and morphologically distinct insoluble tau aggregates in human AD tissue. Furthermore, these research strategies will open the door to compare tau oligomer PTM heterogeneity across several tauopathies, including Cortical basal degeneration and Pick’s disease. Furthermore, the research strategies developed herein can be applied to study the PTM profiles of other aggregation-prone proteins, including α-synuclein, huntingtin, and others.