Dityrosine Cross-links are Present in Alzheimer's Disease-derived Tau Oligomers and Paired Helical Filaments (PHF) which Promotes the Stability of the PHF-core Tau (297-391) In Vitro.

A characteristic hallmark of Alzheimer's Disease (AD) is the pathological aggregation and deposition of tau into paired helical filaments (PHF) in neurofibrillary tangles (NFTs). Oxidative stress is an early event during AD pathogenesis and is associated with tau-mediated AD pathology. Oxidative environments can result in the formation of covalent dityrosine crosslinks that can increase protein stability and insolubility. Dityrosine cross-linking has been shown in Aß plaques in AD and α-synuclein aggregates in Lewy bodies in ex vivo tissue sections, and this modification may increase the insolubility of these aggregates and their resistance to degradation. Using the PHF-core tau fragment (residues 297 - 391) as a model, we have previously demonstrated that dityrosine formation traps tau assemblies to reduce further elongation. However, it is unknown whether dityrosine crosslinks are found in tau deposits in vivo in AD and its relevance to disease mechanism is unclear. Here, using transmission electron microscope (TEM) double immunogold-labelling, we reveal that neurofibrillary NFTs in AD are heavily decorated with dityrosine crosslinks alongside tau. Single immunogold-labelling TEM and fluorescence spectroscopy revealed the presence of dityrosine on AD brain-derived tau oligomers and fibrils. Using the tau (297-391) PHF-core fragment as a model, we further showed that prefibrillar tau species are more amenable to dityrosine crosslinking than tau fibrils. Dityrosine formation results in heat and SDS stability of oxidised prefibrillar and fibrillar tau assemblies. This finding has implications for understanding the mechanism governing the insolubility and toxicity of tau assemblies in vivo.

Paired Helical Filament-Forming Region of Tau (297-391) Influences Endogenous Tau Protein and Accumulates in Acidic Compartments in Human Neuronal Cells.

Assembly of tau protein into paired helical filaments and straight filaments is a key feature of Alzheimer's disease. Aggregation of tau has been implicated in neurodegeneration, cellular toxicity and the propagation, which accompanies disease progression. We have reported previously that a region of tau (297-391), referred to as dGAE, assembles spontaneously in physiological conditions to form paired helical filament-like fibres in vitro in the absence of additives such as heparin. This provides a valuable tool with which to explore the effects of tau in cell culture. Here we have studied the cellular uptake of soluble oligomeric and fibrillar forms of dGAE and examined the downstream consequences of tau internalisation into differentiated SH-SY5Y neuroblastoma cells using fluorescence and electron microscopy alongside structural and biochemical analyses. The assembled dGAE shows more acute cytotoxicity than the soluble, non-aggregated form. Conversely, the soluble form is much more readily internalised and, once within the cell, is able to associate with endogenous tau resulting in increased phosphorylation and aggregation of endogenous tau, which accumulates in lysosomal/endosomal compartments. It appears that soluble oligomeric forms are able to propagate tau pathology without being acutely toxic. The model system we have developed now permits the molecular mechanisms of propagation of tau pathology to be studied in vitro in a more physiological manner with a view to development of novel therapeutic approaches.