Abstract
Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions.Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component (< 0.1%) of insoluble Tau.Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.
Highlights
Intraneuronal inclusions made of filamentous aggregates of the microtubule-associated protein Tau are hallmarks of Alzheimer disease (AD) and related neurodegenerative diseases termed tauopathies, which include progressive supranuclear palsy, corticobasal degeneration and frontotemporal lobe dementia
Using in-gel digestion with trypsin followed by nontargeted LC-MS/MS analysis of Tau bands, we identified five semi-tryptic peptides that could be assigned to LGMN cleavage after four asparagine residues: N167 in the mid-domain of Tau, N255 in the R1 repeat, N296 in the R2 repeat, and N368 at the end of the R4 repeat (Fig. 1a)
Using quantitative western blot of pro-LGMN and activated LGMN, we found that in total homogenate, the ratios of activated-LGMN over pro-LGMN were similar in AD and control hippocampus (Fig. 7), indicating that LGMN activity is not altered in AD brain
Summary
Intraneuronal inclusions made of filamentous aggregates of the microtubule-associated protein Tau are hallmarks of Alzheimer disease (AD) and related neurodegenerative diseases termed tauopathies, which include progressive supranuclear palsy, corticobasal degeneration and frontotemporal lobe dementia. In AD, the formation of Tau inclusions progresses with a typical anatomical pattern as demonstrated by the seminal neuropathological work of Braak and Braak [7] that provide the framework for severity staging. In presymptomatic stages of the disease (Stage I and II), Tau inclusions appear in the entorhinal cortex and hippocampus. During stage III and IV, Tau pathology spreads to the occipito-temporal and insular cortices. At Braak stage V-VI, Tau inclusions propagate throughout the entire cortex [7]. Subtle clinical manifestations or mild cognitive impairment may occur at Braak stage III-IV. AD is clinically symptomatic at Braak stage V-VI
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