Screening of Tau Protein Kinase Inhibitors in a Tauopathy-relevant cell-based model of Tau Hyperphosphorylation and Oligomerization

Abstract

26Tauopathies are a class of neurodegenerative disorders characterized by abnormal deposition of 27 post-translationally modified tau protein in the human brain. Tauopathies are associated with 28 Alzheimer's disease (AD), chronic traumatic encephalopathy (CTE), and other diseases. 29Hyperphosphorylation increases tau tendency to aggregate and forms neurofibrillary tangles 30 (NFT), a pathological hallmark of AD. In this study, okadaic acid (OA, 100 nM), a protein 31 phosphatase 1/2A inhibitor, was treated for 24h in mouse neuroblastoma (N2a) and differentiated 32 rat primary neuronal cortical cell cultures (CTX) to induce tau-hyperphosphorylation and 33 oligomerization as a cell-based tauopathy model. Following the treatments, the effectiveness of 34 different kinase inhibitors was assessed using the tauopathy-relevant tau antibodies through tau-35 immunoblotting, including the sites: pSer202/pThr205 (AT8), pThr181 (AT270), pSer202 (CP13), 36 pSer396/pSer404 (PHF-1), and pThr231 (RZ3). OA-treated samples induced tau phosphorylation 37 and oligomerization at all tested epitopes, forming a monomeric band (46-67 kDa) and oligomeric 38 bands (170 kDa and 240 kDa). We found that TBB (a casein kinase II inhibitor), AR and LiCl 39 (GSK-3 inhibitors), cyclosporin A (calcineurin inhibitor), and Saracatinib (Fyn kinase inhibitor) 40 caused robust inhibition of OA-induced monomeric and oligomeric p-tau in both N2a and CTX 41 culture. Additionally, a cyclin-dependent kinase 5 inhibitor (Roscovitine) and a calcium chelator 42 (EGTA) showed conflicting results between the two neuronal cultures.This study provides a 43 comprehensive view of potential drug candidates (TBB, CsA, AR, and Saracatinib), and their 44 efficacy against tau hyperphosphorylation and oligomerization processes. These findings warrant 45 further experimentation, possibly including animal models of tauopathies, which may provide a 46 putative Neurotherapy for AD, CTE, and other forms of tauopathy-induced neurodegenerative 47 diseases.48

Shaggy functions downstream of dMyc and their concurrent downregulation confers additive rescue against tau toxicity in Drosophila

“…20,21 In our subsequent study, we wanted to investigate the further mechanistic insights of the dmyc mediated rescue event and to identify a cellular connection between dmyc and tau protein. Since kinases are the major controller of the tau etiology 34 and shaggy (gsk3β) being one of the potent tau phosphorylating kinase, we performed a brief genetic screening study to examine any potential association between dmyc and shaggy in tau-mediated disease pathogenesis. Our initial studies indicated that shaggy emerges as a potential interactor that appeared to be working with dmyc in instigating rescue against tau toxicity.…”

Future Therapeutic Perspectives into the Alzheimer’s Disease Targeting the Oxidative Stress Hypothesis

“…All inhibitors showed strong effects toward the kinases investigated, with highlights on gsk-3β in nanomolar ranges [100]. Note that the tau protein has become an increasingly important target in drug discovery, contributing to the development of more efficient therapies for the AD treatment [101,102].…”