Phosphorylated tau interactome in the human Alzheimer's disease brain

Abstract

Accumulation of phosphorylated tau is a key pathological feature of Alzheimer's disease. Phosphorylated tau accumulation causes synaptic impairment, neuronal dysfunction and formation of neurofibrillary tangles. The pathological actions of phosphorylated tau are mediated by surrounding neuronal proteins; however, a comprehensive understanding of the proteins that phosphorylated tau interacts with in Alzheimer's disease is surprisingly limited. Therefore, the aim of this study was to determine the phosphorylated tau interactome. To this end, we used two complementary proteomics approaches: (i) quantitative proteomics was performed on neurofibrillary tangles microdissected from patients with advanced Alzheimer's disease; and (ii) affinity purification-mass spectrometry was used to identify which of these proteins specifically bound to phosphorylated tau. We identified 542 proteins in neurofibrillary tangles. This included the abundant detection of many proteins known to be present in neurofibrillary tangles such as tau, ubiquitin, neurofilament proteins and apolipoprotein E. Affinity purification-mass spectrometry confirmed that 75 proteins present in neurofibrillary tangles interacted with PHF1-immunoreactive phosphorylated tau. Twenty-nine of these proteins have been previously associated with phosphorylated tau, therefore validating our proteomic approach. More importantly, 34 proteins had previously been associated with total tau, but not yet linked directly to phosphorylated tau (e.g. synaptic protein VAMP2, vacuolar-ATPase subunit ATP6V0D1); therefore, we provide new evidence that they directly interact with phosphorylated tau in Alzheimer's disease. In addition, we also identified 12 novel proteins, not previously known to be physiologically or pathologically associated with tau (e.g. RNA binding protein HNRNPA1). Network analysis showed that the phosphorylated tau interactome was enriched in proteins involved in the protein ubiquitination pathway and phagosome maturation. Importantly, we were able to pinpoint specific proteins that phosphorylated tau interacts with in these pathways for the first time, therefore providing novel potential pathogenic mechanisms that can be explored in future studies. Combined, our results reveal new potential drug targets for the treatment of tauopathies and provide insight into how phosphorylated tau mediates its toxicity in Alzheimer's disease.

Keywords: Alzheimer’s disease; neurofibrillary tangles; phosphorylation; proteomics; tau.

Figure 1

Protein families that were most enriched in NFTs in advanced Alzheimer’s disease. Enrichment of protein families was determined using PFAM enrichment analysis within STRING. The protein families that were most significantly enriched in NFTs are shown.

Figure 2

Phosphorylated residues identified to be present on tau by AP-MS. Numbering corresponds to the longest tau isoform (441 amino acids in length). Phosphorylated residues recognized by PHF1 are shown in red.

Figure 3

Proteins identified by AP-MS for pTau. Each point corresponds to an individual protein plotted by fold change difference after co-IP for pTau versus isotype control antibody (x-axis) and the probability that a protein is a pTau interactor (SAINT score; y-axis). SAINT score = 1 identifies proteins with the highest probability of being a pTau interactor (proteins with a SAINT score = 1 detailed in the box labelled ‘most significant pTau interactors’). One hundred and twenty-five proteins were found to be significant pTau interactors (highlighted in red). Enriched pathways/families highlights examples of the most significantly enriched protein pathways or families identified by enrichment analysis.

Figure 4

Overlap of pTau interactors and proteins found in NFTs. Seventy-five proteins present in NFTs were also significant pTau interactors. Overlapping proteins are shown by gene IDs, with the font size corresponding to the number of peptide spectrum matches for each protein after pTau AP-MS. As expected, tau (MAPT) was the most abundant protein identified after AP-MS, followed by ubiquitin (UBB).

Figure 5

Validation of AP-MS results using co-IP. Validation of HSP90B1, SCRN1 (positive control) and EZR (negative control) interactions with pTau. Co-IP was performed on fresh frozen frontal cortex tissue from one Alzheimer’s disease case. Immunoprecipitation was performed using anti-HSP90B1, anti-SCRN1, anti-EZR, rabbit IgG isotype control or mouse IgG isotype control. Both HSP90B1 and SCRN1 pulled down pTau, while EZR did not. Presence of pTau, HPS90B1, SCRN1 and EZR in input brain homogenate was confirmed. Asterisks indicate bands corresponding to cross-reactive IgGs.