No abstract

Tau aggregation and accumulation is a key event in the pathogenesis of Alzheimer's disease. Inhibition of Tau aggregation is therefore a potential therapeutic strategy to ameliorate the disease. Phytochemicals are being highlighted as potential aggregation inhibitors. Epigallocatechin-3-gallate (EGCG) is an active phytochemical of green tea that has shown its potency against various diseases including aggregation inhibition of repeat Tau. The potency of EGCG in altering the PHF assembly of full-length human Tau has not been fully explored. By various biophysical and biochemical analyses like ThS fluorescence assay, MALDI-TOF analysis and Isothermal Titration Calorimetry, we demonstrate dual effect of EGCG on aggregation inhibition and disassembly of full-length Tau and their binding affinity. The IC50 for Tau aggregation by EGCG was found to be 64.2 μM. Abbreviations EGCG Epigallocatechin-3-gallate AD Alzheimer's disease Aβ Amyloid-β IAPP Islet amyloid polypeptide ITC Isothermal titration calorimetry TEM Transmission electron microscopy SEC Size exclusion chromatography MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ThS Thioflavin S ANS 8-Anilino-1-naphthalenesulfonic acid ammonium salt The direct causal relationship between neurodegenerative disorders and protein misfolding has been debatable over years 1-3. The recent advances in studies of misfolded proteins to some extent have shed the light upon the cause-effect interdependence of proteins and diseases 4. Neurodegenerative diseases are diverse in nature at physiological as well as molecular level. For example, the misfolded proteins involved in Alzheimer's disease (AD) are amyloid-β (Aβ) and Tau (Fig. 1A) whereas 5 ; α-Synuclein is a key player in Parkinson's disease. AD is phenotypically characterized by gradual memory loss due to neuronal death 6. The molecular pathology involves abnormal accumulation of Aβ plaques 7 in the extracellular milieu and cytoplasmic Tau tangles 8,9. Tau aggregation increases load on clearance machinery of neurons, which finally collapses, and results in neuronal death 10,11. The physiological function of Tau is to bind microtubules and stabilize them thus aiding in neuronal functioning 12,13. Tau aggregates abnormally due to multiple factors-like mutations, aberrant post-translational modifications, oxidative stress etc. 14-16. Abnormal phosphorylation of Tau is one of the key factors implied in its aggregation 17,18. The factors leading to Tau aggregation have directed the researchers to design and develop the therapeutics against Tau aggregation and AD. Several classes of compounds showing potency in inhibiting Tau aggregation include phenothiazines 19,20 , anthraquinones 21 , porphyrins, aminothienopyridazines 22 , natural compounds (polyphenols 23 , secondary metabolites 24 , curcumin 25 , Oleocanthal 26 etc.). The phenothiazine compounds inhibit Tau-Tau (repeat domain) binding thus preventing Tau aggregation. These compounds have

A native conformation of a protein is essential for its biological role. In certain conditions, some proteins show non-native conformations, leading to aggregation, which in turn may produce severe pathologies. Such physiological conditions are classified as protein misfolding diseases. Alzheimer's disease (AD) is the most common form of dementia. Extracellular senile plaques formed by Amyloid β and intracellular aggregates formed by microtubule-associated protein Tau (MAPT) are the hallmarks of AD. Physiological role of MAPT is to maintain the integrity and stability of microtubules, however it tends to self-aggregate forming intracellular paired helical filaments (PHFs) during AD. MAPT is also subjected to various post-translational modifications such as phosphorylation, glycosylation, truncation, and acetylation. Being natively unfolded, MAPT is prone to full characterization at atomic level. Small-angle X-ray scattering (SAXS) is often applied in combination with other biophysical methods, like nuclear magnetic resonance (NMR), circular dichroism (CD), fluorescence spectroscopy, analytical ultracentrifugation (AUC), or dynamic light scattering (DLS) to characterize natively unfolded systems. Here we describe the practical aspects of MAPT characterization by SAXS and CD in detail as well as outline the inferred structural and functional implications.