Publication: MOLECULAR MECHANISM OF NOVEL ANTI-PHOSPHO-TAU SERINE 396/404 SINGLE CHAIN VARIABLE FRAGMENT (scFv) ANTIBODY ON THE REVERSAL OF TAU PATHOLOGY IN ALZHEIMER’S DISEASE
Date
2023
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Publisher
International Medical University
Abstract
Alzheimer’s disease (AD) is an aging-associated neurodegenerative disorder which produces a progressive and drastic decline in memory and learning. Currently, there are no effective treatment for AD. The major pathological hallmarks of AD are intracellular neurofibrillary tangles (NFTs) formation and amyloid-β (Aβ) plaques deposition in the brain. However, Aβ-dedicated therapies were not effective at treating AD as the efficacy of Aβ therapies observed in animal models is not reflected in human clinical trials. Therefore, therapies targeting Tau have gained tremendous attention as the potential treatments for AD. Previous studies had shown that antibody against phospho-Tau serine 396/404 could prevent Tau aggregation and clearing Tau oligomers and insoluble aggregates. In this study, anti-phospho-Tau serine 396/404 single chain variable fragment (scFv) antibody was generated by using Ph.D-12 phage display library. In this process, four rounds of biopanning and amplifications were carried out. Furthermore, phage ELISA binding assay was conducted to determine binding affinity of phage in each round towards the epitope. The results indicate the phage in fourth round are highly specific towards the epitope. After fourth round of biopanning and amplification, three scFv antibodies which appeared as peptide sequence motif, namely FPLNSEENPFEL, FPLNSEENPLEL and FPLNSEENAFEL were identified. These scFv antibodies were later tested on immortalised hepatocyte, CHANG, to investigate their toxicity towards normal cells via MTT cell viability assay. The results were indicating scFvs were not toxic towards immortalised hepatocytes. The scFvs antibodies also treated on AD cell model for their efficacy in eliminating hyperphosphorylated Tau. The AD cell model (T-SH-SY5Y) was produced by transfecting neuroblastoma cells, SH-SY5Y, with 2N4R Tau-441 plasmid. A protein phosphatase inhibitor, okadaic acid (OA), was added to T-SH-SY5Y cells to induce hyperphosphorylation of Tau protein. Based on MTT cell viability results, the cell viabilities had increased after treated with 10 μM scFvs antibodies for 48 hours compared to those treated with 20 nM OA. Moreover, the pSer396 and PHF-1 expression level was also decreased after treated T-SH-SY5Y cells with 10 μM scFv antibodies. The results indicated the cytotoxic effect of hyperphosphorylated Tau was reduced or eradicated by treating the T-SH-SY5Y cells with scFv antibodies. Molecular mechanism of scFv antibodies on elimination of hyperphosphorylated Tau was investigated in this study. Autophagy is a major self-degenerative process to maintain cellular homeostasis and function. Phosphatidylinositol 3-phosphate kinase (PI3K)/AKT/mammalian target of rapamycin (m-TOR) signalling pathway is the primary pathway that regulates autophagy when cells are under certain conditions such as starvation and oxidative stress. There is vast evidence have related PI3K/AKT/m-TOR signalling pathway to AD especially Tau phosphorylation. Overall, the protein expression of PI3K, AKT and m-TOR were decreased in 10 μM scFv antibodies treated T-SH-SY5Y cells. Numerous research had claimed that downregulation for these three proteins could induced autophagy in cells. A regulator at upstream of PI3K/AKT/m-TOR signalling pathway, PTEN, was also study by using western blot analysis. The results shown that PTEN protein expression was increased scFv antibodies treated T-SH-SY5Y cells. Upregulation of PTEN protein was known to inhibit the proteins (PI3K, AKT and m-TOR) involved in downstream of the signalling pathway which ultimately induced autophagy. Hence, the scFv antibodies able to eradicate Tau hyperphosphorylation via PI3K/AKT/m-TOR signalling pathway by inducing autophagy in AD cell model. In this study, possibilities of hyperphosphorylated Tau elimination via endoplasmic reticulum (ER) stress signalling pathway in T-SH-SY5Y cells also investigated. Several studies had reported ER-stress and Tau hyperphosphorylation could be induced by each other which led to promote AD-like neurodegeneration. Protein expression level of selected proteins (IRE-1-α, Calnexin, PERK, Atg12, Beclin-1 and eIF-2α) which involved in ER-stress pathway were studies by using western blot analysis. Based on western blot analysis results, majority of the protein expression level of selected protein was decreased by treating T-SH-SY5Y cells with scFv antibodies. However, the expression level of selected protein was increased in T-SH-SY5Y treated with scFvs and its mixture for 3 hours. The results indicated the ER-stress pathway was activated in early events of scFv treatment which led to autophagy in AD cell model. In conclusion, three scFv antibodies with high affinity towards phospho-Tau serine 396/404 epitope was identified. All of them did not induced cytotoxicity on immortalised hepatocytes and able to reduce cytotoxicity of hyperphosphorylated Tau protein in AD cell model. The eradication of Tau hyperphosphorylation by these scFv antibodies can be achieved via inhibition of PI3K/AKT/m-TOR and ER-stress signalling pathway.
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Keywords
Alzheimer Disease, Neurodegenerative Diseases, Hepatocytes, Phosphoprotein Phosphatases, Autophagy