Despite a key role of amyloid-beta (Aβ) in Alzheimer's disease (AD), mechanisms that link Aβ plaques to tau neurofibrillary tangles and cognitive decline still remain poorly understood. The purpose of this study was to quantify proteins in the sarkosyl-insoluble brain proteome correlated with Aβ and tau insolubility in the asymptomatic phase of AD (AsymAD) and through mild cognitive impairment (MCI) and symptomatic AD. Employing label-free mass spectrometry-based proteomics, we quantified 2711 sarkosyl-insoluble proteins across the prefrontal cortex from 35 individual cases representing control, AsymAD, MCI and AD. Significant enrichment of Aβ and tau in AD was observed, which correlated with neuropathological measurements of plaque and tau tangle density, respectively. Pairwise correlation coefficients were also determined for all quantified proteins to Aβ and tau, across the 35 cases. Notably, six of the ten most correlated proteins to Aβ were U1 small nuclear ribonucleoproteins (U1 snRNPs). Three of these U1 snRNPs (U1A, SmD and U1-70K) also correlated with tau consistent with their association with tangle pathology in AD. Thus, proteins that cross-correlate with both Aβ and tau, including specific U1 snRNPs, may have potential mechanistic roles in linking Aβ plaques to tau tangle pathology during AD progression.
Several neurodegenerative diseases including Alzheimer's Disease (AD) are characterized by ubiquitin-positive pathological protein aggregates. Here, an immunoaffinity approach is utilized to enrich ubiquitylated isopeptides after trypsin digestion from five AD and five age-matched control postmortem brain tissues. Label-free MS-based proteomic analysis identifies 4291 unique ubiquitylation sites mapping to 1682 unique proteins. Differential enrichment analysis shows that over 800 ubiquitylation sites are significantly altered between AD and control cases. Of these, ≈80% are increased in AD, including seven poly ubiquitin linkages, which is consistent with proteolytic stress and high burden of ubiquitylated pathological aggregates in AD. The microtubule associated protein Tau, the core component of neurofibrillary tangles, has the highest number of increased sites of ubiquitylation per any protein in AD. Tau poly ubiquitylation from AD brain homogenates is confirmed by reciprocal co-immunoprecipitation and by affinity capture using tandem ubiquitin binding entities. Co-modified peptides, with both ubiquitylation and phosphorylation sites, are also enriched in AD. Notably, many of the co-modified peptides mapped to Tau within KXGS motifs in the microtubule binding region suggesting that crosstalk between phosphorylation and ubiquitylation occurs on Tau in AD. Overall, these findings highlight the utility of MS to map ubiquitylated substrates in human brain and provides insight into mechanisms underlying pathological protein posttranslational modification in AD.
Background
Detergent-insoluble protein accumulation and aggregation in the brain is one of the pathological hallmarks of neurodegenerative diseases. Here, we describe the identification of septin 11 (SEPT11), an enriched component of detergent-resistant fractions in frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (FTLD-U), using large-scale unbiased proteomics approaches.
Results
We developed and applied orthogonal quantitative proteomic strategies for the unbiased identification of disease-associated proteins in FTLD-U. Using these approaches, we proteomically profiled detergent-insoluble protein extracts prepared from frontal cortex of FTLD-U cases, unaffected controls, or neurologic controls (i.e. Alzheimer's disease; AD). Among the proteins altered specifically in FTLD-U, we identified TAR DNA binding protein-43 (TDP-43), a known component of ubiquitinated inclusions. Moreover, we identified additional proteins enriched in detergent-resistant fractions in FTLD-U, and characterized one of them, SEPT11, in detail. Using independent highly sensitive targeted proteomics approaches, we confirmed the enrichment of SEPT11 in FTLD-U extracts. We further showed that SEPT11 is proteolytically cleaved into N-terminal fragments and, in addition to its prominent glial localization in normal brain, accumulates in thread-like pathology in affected cortex of FTLD-U patients.
Conclusions
The proteomic discovery of insoluble SEPT11 accumulation in FTLD-U, along with novel pathological associations, highlights a role for this cytoskeleton-associated protein in the pathogenesis of this complex disorder.