Novel Peptidomic Approach for Identification of Low and High Molecular Weight Tauopathy Peptides Following Calpain Digestion, and Primary Culture Neurotoxic Challenges.

Hamad Yadikar,Firas Kobeissy,Isabel Torres,Milin Kurup,Edwin Mouhawasse,Kevin K Wang,Richard Yost,Niko Pafundi,Connor Johnson,Zhihui Yang,Lynn Nguyen

doi:10.3390/ijms20205213

Abstract

Tauopathy is a class of a neurodegenerative disorder linked with tau hyperphosphorylation, proteolysis, and aggregation. Tau can be subjected to proteolysis upon calpain activation in Alzheimer disease (AD), and traumatic brain injury (TBI). We and others have extensively researched calpain-mediated tau breakdown products (Tau-BDP; 45K, 35K, and 17K). Tau proteolysis might also generate low molecular weight (LMW ≤10K) proteolytic peptides after neurodegenerative damage. In this study, we have subjected purified tau protein (phospho and non-phospho) and mouse brain lysate to calpain-1 digestion to characterize the LMW generated by nano-liquid chromatography coupled to electrospray ionization to tandem mass spectrometry (nano-LC-ESI-MS/MS). We have also challenged differentiated primary cerebrocortical neuronal cultures (CTX) with neurotoxic agents (calcium ionophore calcimycin (A23187), staurosporine (STS), N-methyl-D-aspartate (NMDA), and Maitotoxin (MTX)) that mimic neurodegeneration to investigate the peptidome released into the conditioned cell media. We used a simple workflow in which we fractionate LMW calpain-mediated tau peptides by ultrafiltration (molecular weight cut-off value (MWCO) of 10K) and subject filtrate fractions to nano-LC-MS/MS analysis. The high molecular weight (HMW) peptides and intact proteins retained on the filter were analyzed separately by western blotting using total and phospho-specific tau antibodies. We have identified several novel proteolytic tau peptides (phosphorylated and non-phosphorylated) that are only present in samples treated with calpain or cell-based calpain activation model (particularly N- and C-terminal peptides). Our findings can help in developing future research strategies emphasizing on the suppression of tau proteolysis as a target.

Highlights

Microtubule-associated protein tau (MAPT) is a soluble protein whose function is to control the stability of the axonal microtubules [1]
We have included the non-phosphorylated and phosphorylated purified tau-441 (p-tau) protein in our study to account for the differential proteolytic peptides that can be generated by calpain digestion
The neurotoxic agents were used (staurosporine (STS), calcium ionophore (A23187), and maitotoxin (MTX)) on a differentiated rat primary cerebrocortical neuronal culture in the presence and absence of okadaic acid (OA) (Figure 1C) to characterize the proteolytic peptides generated under tauopathy neurodegenerative conditions [22,23,24]

Summary

Introduction

Microtubule-associated protein tau (MAPT) is a soluble protein whose function is to control the stability of the axonal microtubules [1]. Tauopathies consist of abnormal post-translational modifications of tau that leads to the formation of tau oligomers, pair helical filaments, and neurofibrillary tangles. Tauopathies of the CNS, including Alzheimer Disease (AD), chronic traumatic encephalopathy (CTE), and Parkinson’s disease, are associated with defective tau protein that can no longer stabilize the microtubule [2]. Processes occurring in tauopathies disrupt tau physiological function of microtubule stabilization and formation [3,4,5]. Examples of post-translational modifications (PTMs) that occur in tauopathies include hyperphosphorylation, acetylation, deamination, glycation, glycosylation, isomerization, methylation, nitration sumoylation, ubiquitination, and proteolysis [6]. Phosphorylation is one of the most well-characterized and researched PTM of tau, for its direct ability to disrupt binding to microtubules and contribution to aggregation, as well as for neurofibrillary tangles (NFT) formation [7]

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