Interaction of Tau with G-Protein-Coupled Purinergic P2Y12 Receptor by Molecular Docking and Molecular Dynamic Simulation.

Alzheimer's disease, a progressive neurological disorder, is characterized by the accumulation of neurofibrillary tangles and senile plaques by Tau and amyloid-ß, respectively, in the brain microenvironment. The misfolded protein aggregates interact with several components of neuronal and glial cells such as membrane lipids, receptors, transporters, enzymes, cytoskeletal proteins, etc. Under pathological conditions, Tau interacts with several G-protein-coupled receptors (GPCRs), which undergoes either receptor signaling or desensitization followed by internalization of the protein complex. The purinergic GPCR, P2Y12 which is expressed in microglial cells, plays a key role in its activation and migration. Microglial cells sense and migrate to the site of injury aided by P2Y12 receptor that interacts with ADP released from damaged cells. P2Y12 receptor also interacts with misfolded Tau accumulated at the extracellular space and promotes receptor-mediated internalization. Immunocolocalization and co-immunoprecipitation studies demonstrated the interaction of Tau species with the P2Y12 receptor. Later, in-silico analyses were carried out with the repeat domain of Tau (TauRD), which has been identified as the interacting partner of P2Y12 receptor by in-vitro studies. Molecular docking and molecular dynamics simulation studies show the stability and the type of interaction in TauRD-receptor complex. Tau interaction with P2Y12 receptor plays a significant role in maintaining the active state of microglia which could lead to neuroinflammation and neuronal damage in AD brain. Hence, blocking P2Y12-Tau interaction and P2Y12-mediated Tau internalization in microglial cells could be possible therapeutic strategies in downregulating the severity of neuroinflammation in AD.

Biochemical and Biophysical Characterization of Tau and α-Linolenic Acid Vesicles In Vitro.

Alzheimer's disease (AD) is characterized by the abnormal accumulation of disordered protein, that is, extracellular senile plaques of amyloid-ß (Aß) and intracellular neurofibrillary tangles of Tau. Tau protein has gained the attention in recent years owing to the ability to propagate in a "prion-like" nature. The disordered protein Tau possesses a high positive charge, which allows its binding to anionic proteins and factors. The native disorder of proteins attends the ß-sheet structure from its random-coiled conformation upon charge compensation by various polyanionic agents such as heparin, RNA, etc. Anionic lipids such as arachidonic acid (AA) and oleic acid (OA) are also one of the factors which can induce aggregation of Tau in physiological conditions. The free units of Tau protein can bind to lipid membranes through its repeat domain (RD), the anionic side chains of the membrane lipids induce aggregation of Tau by reducing the activation barrier. In this study, we investigated the role of α-linolenic acid (ALA) as an inducing agent for Tau aggregation in vitro conditions. Omega-3 fatty acids bear a capacity to reduce the pathology of Tau by downregulating the Tau phosphorylation pathway. We have studied by using various biochemical or biophysical methods the potency of ALA as an aggregating agent for Tau. We have implemented different techniques such as SDS-PAGE, transmission electron microscopy, CD spectroscopy to evaluated higher-order aggregates of Tau upon induction by ALA.

Purinergic Receptor P2Y12-Mediated Tau Internalization in Microglia.

Microglia are the resident brain macrophage cells that are involved in constant surveillance of brain microenvironment. In Alzheimer's disease, microglia get over activated upon the accumulation of Tau and amyloid-ß species in the extracellular space, ultimately leading to neurodegeneration. Microglia phagocytose the extracellular Tau species by several mechanisms among which P2Y12 receptor-mediated internalization of extracellular Tau is recently studied. Extracellular Tau activates microglia and directly interacts with the P2Y12 receptor. Tau-receptor complex is then internalized followed by perinuclear accumulation and lysosomal degradation. Upon microglial activation by extracellular Tau, P2Y12 receptor is also involved in membrane-associated actin remodeling which has its key role in active migration and phagocytosis.

α-Linolenic Acid Induces Microglial Activation and Extracellular Tau Internalization.

Neuroinflammation is the brain condition that occurs due to the hyper-activation of brain's immune cells and microglia, over the stimulation of extracellular aggregated proteins such as amyloid plaques and by extracellular Tau as well. The phenotypic changes of microglia from inflammatory to anti-inflammatory can be triggered by many factors, which also includes dietary fatty acids. The classes of omega-3 fatty acids are the majorly responsible in maintaining the anti-inflammatory phenotype of microglia. The enhanced phagocytic ability of microglia might induce the clearance of extracellular aggregated proteins, such as amyloid beta and Tau. In this study, we emphasized on the effect of α-linolenic acid (ALA) on the activation of microglia and internalization of the extracellular Tau seed in microglia.

Microglial Uptake of Extracellular Tau by Actin-Mediated Phagocytosis.

Microglia are scavengers of the brain environment that clear dead cells, debris, and microbes. In Alzheimer's disease, microglia get activated to phagocytose damaged neurons, extracellular Amyoid-ß, and Tau deposits. Several Tau internalization mechanisms of microglia have been studied which include phagocytosis, pinocytosis, and receptor-mediated endocytosis. In this chapter, we have visualized microglial phagocytic structures that are actin-rich cup-like extensions, which surrounds extracellular Tau species by wide-field fluorescence and confocal microscopy. We have shown the association of filamentous actin in Tau phagocytosis along the assembly of LC-3 molecules to phagosomes. The 3-dimensional, orthogonal and gallery wise representation of these phagocytic structures provides an overview of the phagocytic mechanism of extracellular Tau by microglia.

Internalization and Endosomal Trafficking of Extracellular Tau in Microglia Improved by α-Linolenic Acid.

Alzheimer's disease (AD) is distinguished by extracellular accumulation of amyloid-beta plaques and intracellular neurofibrillary tangles of Tau. Pathogenic Tau species are also known to display "prion-like propagation," which explains their presence in extracellular spaces as well. Glial population, especially microglia, tend to proclaim neuroinflammatory condition, disrupted signaling mechanisms, and cytoskeleton deregulation in AD. Omega-3 fatty acids play a neuroprotective role in the brain, which can trigger the anti-inflammatory pathways as well as actin dynamics in the cells. Improvement of cytoskeletal assembly mechanism by omega-3 fatty acids would regulate the other signaling cascades in the cells, leading to refining clearance of extracellular protein burden in AD. In this study, we focused on analyzing the ability of α-linolenic acid (ALA) as a regulator of actin dynamics to balance the signaling pathways in microglia, including endocytosis of extracellular Tau burden in AD.

Understanding Actin Remodeling in Neuronal Cells Through Podosomes.

Cytoskeletal dysregulation forms an important aspect of many neurodegenerative diseases such as Alzheimer's disease. Cytoskeletal functions require the dynamic activity of the cytoskeletal proteins-actin, tubulin, and the associated proteins. One of such important phenomena is that of actin remodeling, which helps the cell to migrate, navigate, and interact with extracellular materials. Podosomes are complex actin-rich cytoskeletal structures, abundant in proteins that interact and degrade the extracellular matrix, enabling cells to displace and migrate. The formation of podosomes requires extensive actin networks and remodeling. Here we present a novel immunofluorescence-based approach to study actin remodeling in neurons through the medium of podosomes.

G-protein coupled purinergic P2Y12 receptor interacts and internalizes TauRD-mediated by membrane-associated actin cytoskeleton remodeling in microglia.

In Alzheimer's disease, the microtubule-associated protein, Tau misfolds to form aggregates and filaments in the intra- and extracellular region of neuronal cells. Microglial cells are the resident brain macrophage cells involved in constant surveillance and activated by the extracellular deposits. Purinergic receptors are involved in the chemotactic migration of microglial cells towards the site of inflammation. From our recent study, we have observed that the microglial P2Y12 receptor is involved in phagocytosis of full-length Tau species such as monomers, oligomers and aggregates by actin-driven chemotaxis. This study shows the interaction of repeat-domain of Tau (TauRD) with the microglial P2Y12 receptor and the corresponding residues for interaction have been analysed by various in-silico approaches. In the cellular studies, TauRD was found to interact with microglial P2Y12R and induces its cellular expression confirmed by co-immunoprecipitation and western blot analysis. Furthermore, the P2Y12R-mediated TauRD internalization has demonstrated activation of microglia with an increase in the Iba1 level, and TauRD becomes accumulated at the peri-nuclear region for the degradation. Similarly, immunofluorescence microscopic studies emphasized that TauRD is phagocytosed by microglial P2Y12R via the membrane-associated actin remodeling as filopodia extension. Upon internalization, we have demonstrated the P2Y12R signaling-mediated degradation of accumulated TauRD by lysosomal pathway. Altogether, microglial P2Y12R interacts with TauRD and mediates directed migration and activation for its internalization and degradation.

Role of cysteines in accelerating Tau filament formation.

Alzheimer's disease is majorly associated with intracellular accumulation of Tau into paired helical filaments and tangles. The self-aggregated dimeric and oligomeric species of Tau formed are more toxic to neuronal cells and acts as seeds for filament formation. The two cysteine residues and the two hexapeptide regions of full-length Tau play a key role in initialization and filament formation during Tau aggregation. The role of cysteine residues in Tau aggregation has been studied by in-vitro aggregation assay that was measured by Thioflavin S fluorescence to observe the kinetics of aggregation. In this study, we have performed in-vitro aggregation assay with recombinant full-length Tau and the cysteine mutants to understand the mechanism of cysteine independent Tau aggregation. Here, we report that cysteine mutant full-length Tau can aggregate to form filaments under in-vitro conditions. To visualize the polymorphisms of Tau and cysteine mutants under different aggregation conditions anionic cofactor, heparin was employed. Wild-type Tau showed rapid aggregation to form oligomers and filaments. On the other hand, the cysteine mutant delayed the initial Tau aggregation. This indicates the importance of cysteine residues in accelerating initial Tau nucleation for its aggregation. The filament morphology of wild-type and cysteine mutant Tau has been characterized using transmission electron microscopy and high-resolution transmission electron microscopy.Communicated by Ramaswamy H. Sarma.

G-protein coupled receptor, PI3K and Rho signaling pathways regulate the cascades of Tau and amyloid-ß in Alzheimer's disease.

Alzheimer's disease is a progressive neurodegenerative disease characterized by the presence of amyloid-ß plaques in the extracellular environment and aggregates of Tau protein that forms neurofibrillary tangles (NFTs) in neuronal cells. Along with these pathological proteins, the disease shows neuroinflammation, neuronal death, impairment in the immune function of microglia and synaptic loss, which are mediated by several important signaling pathways. The PI3K/Akt-mediated survival-signaling pathway is activated by many receptors such as G-protein coupled receptors (GPCRs), triggering receptor expressed on myeloid cells 2 (TREM2), and lysophosphatidic acid (LPA) receptor. The signaling pathway not only increases the survival of neurons but also regulates inflammation, phagocytosis, cellular protection, Tau phosphorylation and Aß secretion as well. In this review, we focused on receptors, which activate PI3K/Akt pathway and its potential to treat Alzheimer's disease. Among several membrane receptors, GPCRs are the major drug targets for therapy, and GPCR signaling pathways are altered during Alzheimer's disease. Several GPCRs are involved in the pathogenic progression, phosphorylation of Tau protein by activation of various cellular kinases and are involved in the amyloidogenic pathway of amyloid-ß synthesis. Apart from various GPCR signaling pathways, GPCR regulating/ interacting proteins are involved in the pathogenesis of Alzheimer's disease. These include several small GTPases, Ras homolog enriched in brain, GPCR associated sorting proteins, ß-arrestins, etc., that play a critical role in disease progression and has been elaborated in this review.

Role of Microglia in Regulating Cholesterol and Tau Pathology in Alzheimer's Disease.

Cholesterol, a principal constituent of the cell membrane, plays a crucial role in the brain by regulating the synaptic transmission, neuronal signaling, as well as neurodegenerative diseases. Defects in the cholesterol trafficking are associated with enhanced generation of hyperphosphorylated Tau and Amyloid-ß protein. Tau, a major microtubule-associated protein in the brain, is the key regulator of the mature neuron. Abnormally hyperphosphorylated Tau hampers the major functions related to microtubule assembly by promoting neurofibrillary tangles of paired helical filaments, twisted ribbons, and straight filaments. The observed pathological changes due to impaired cholesterol and Tau protein accumulation cause Alzheimer's disease. Thus, in order to regulate the pathogenesis of Alzheimer's disease, regulation of cholesterol metabolism, as well as Tau phosphorylation, is essential. The current review provides an overview of (1) cholesterol synthesis in the brain, neurons, astrocytes, and microglia; (2) the mechanism involved in modulating cholesterol concentration between the astrocytes and brain; (3) major mechanisms involved in the hyperphosphorylation of Tau and amyloid-ß protein; and (4) microglial involvement in its regulation. Thus, the answering key questions will provide an in-depth information on microglia involvement in managing the pathogenesis of cholesterol-modulated hyperphosphorylated Tau protein.

HDAC6 ZnF UBP as the Modifier of Tau Structure and Function.

Histone deacetylase 6 is a class II histone deacetylase primarily present in the cytoplasm and involved in the regulation of various cellular functions. It consists of two catalytic deacetylase domains and a unique zinc finger ubiquitin binding protein domain, which sets it apart from other HDACs. HDAC6 is known to regulate cellular activities by modifying the function of microtubules, HSP90, and cortactin through deacetylation. Apart from the catalytic activity of HDAC6, it interacts with other proteins through either the SE14 domain or the ZnF UBP domain to modulate their functions. Here, we have studied the role of the HDAC6 ZnF UBP domain as a modifier of Tau aggregation by its direct interaction with the polyproline region/repeat region of Tau. Interaction of HDAC6 ZnF UBP with Tau was found to reduce the propensity of Tau to self-aggregate and to disaggregate preformed aggregates in a concentration-dependent manner and also bring about the conformational changes in Tau protein. The interaction of HDAC6 ZnF UBP with Tau results in its degradation, suggesting either proteolytic activity of HDAC6 ZnF UBP or its role in enhancing autoproteolysis of Tau.

Interaction of Tau with the chemokine receptor, CX3CR1 and its effect on microglial activation, migration and proliferation.

Alzheimer's disease (AD) is a neurodegenerative disease that leads to progressive loss of memory and dementia. The pathological hallmarks of AD include extracellular accumulation of amyloid-ß peptides forming senile plaques and intracellular accumulation of Tau oligomers and filamentous species. Tau is a microtubule-binding protein that stabilizes tubulin to form microtubules under physiological condition. In AD/ pathological condition, Tau detaches from microtubules and aggregates to form oligomers of different sizes and filamentous species such as paired helical filaments. Microglia are the resident brain macrophages that are involved in the phagocytosis of microbes, cellular debris, misfolded and aggregated proteins. Chemokine receptor, CX3CR1 is mostly expressed on microglia and is involved in maintaining the microglia in a quiescent state by binding to its ligand, fractalkine (CX3CL1), which is expressed in neurons as both soluble or membrane-bound state. Hence, under physiological conditions, the CX3CR1/CX3CL1 axis plays a significant role in maintaining the central nervous system (CNS) homeostasis. Further, CX3CR1/CX3CL1 signalling is involved in the synthesis of anti-inflammatory cytokines and also has a significant role in cytoskeletal rearrangement, migration, apoptosis and proliferation. In AD brain, the expression level of fractalkine is reduced, and hence Tau competes to interact with its receptor, CX3CR1. In microglia, phagocytosis and internalization of extracellular Tau species occurs in the presence of a chemokine receptor, CX3CR1 which binds directly to Tau and promotes its internalization. In this review, the pathophysiological roles of CX3CR1/fractalkine signalling in microglia and neurons at different stages of Alzheimer's disease and the possible role of CX3CR1/Tau signalling has been widely discussed.

EGCG impedes human Tau aggregation and interacts with Tau.

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.

G-Protein Coupled Receptors and Tau-different Roles in Alzheimer's Disease.

Post-translational modification of Tau, a microtubule-associated protein in the neuronal cell, plays a major role in Alzheimer's disease. Tau is an axonal protein expressed in mature neurons that promote the self-assembly of tubulin into microtubules and its stabilization in neurons. Phosphorylation of Tau makes it prone to aggregation at the intra-neuronal region leading to impaired neurotransmission and dementia. Tau aggregates undergo trans-cellular propagation by the release of fibrillar species into the extra-cellular environment from damaged and infected neurons that can be internalized by neighbouring neuronal and glia cells and promotes aggregation in healthy cells. G-protein coupled receptors, the largest group of seven transmembrane receptors, are involved in neuronal signal transduction in response to various signals such as hormones and neurotransmitters. In Alzheimer's disease, GPCRs are involved in phosphorylation of Tau through various downstream kinases such as GSK-3ß, CDK-5 and ERKs signalling cascade. Several neuronal GPCRs that are involved in Tau phosphorylation are elaborated in this review. The astrocytic GPCR, Tau phosphorylation mediated by CaS receptors and its propagation by exosomes are also elaborated. In the microglia, the extra-cellular Tau binding to a chemokine GPCR, CX3CR1 triggers its internalization, whereas Tau phosphorylation at specific sites decreases its binding affinity to this receptor. Here we highlight the role of GPCRs in Tau phosphorylation and Tau interaction in different cells of the nervous system. Hence, the role of GPCRs are attaining more attention in the therapeutic field of Alzheimer's disease. Specific agonists/antagonists and allosteric modulators could be the potential target for therapy against GPCR-mediated Tau phosphorylation in Alzheimer's disease.

Basic Limonoid modulates Chaperone-mediated Proteostasis and dissolve Tau fibrils.

The Alzheimer's disease pathology is associated with accumulation of intracellular neurofibrillary tangles and extracellular senile plaques. The formation of initial nucleus triggers conformational changes in Tau and leads to its deposition. Hence, there is a need to eliminate these toxic proteins for proper functioning of neuronal cells. In this aspect, we screened the effect of basic limonoids such as gedunin, epoxyazadiradione, azadirone and azadiradione on inhibiting Tau aggregation as well as disintegration of induced Tau aggregates. It was observed that these basic limonoids effectively prevented aggregates formation by Tau and also exhibited the property of destabilizing matured Tau aggregates. The molecular docking analysis suggests that the basic limonoids interact with hexapeptide regions of aggregated Tau. Although these limonoids caused the conformational changes in Tau to ß-sheet structure, the cytological studies indicate that basic limonoids rescued cell death. The dual role of limonoids in Tau aggregation inhibition and disintegration of matured aggregates suggests them to be potent molecules in overcoming Tau pathology. Further, their origin from a medicinally important plant neem, which known to possess remarkable biological activities was also found to play protective role in HEK293T cells. Basic limonoids were non-toxic to HEK293T cells and also aided in activation of HSF1 by inducing its accumulation in nucleus. Western blotting and immunofluorescence studies showed that HSF1 in downstream increased the transcription of Hsp70 thus, aggravating cytosolic Hsp70 levels that can channel clearance of aberrant Tau. All these results mark basic limonoids as potential therapeutic natural products.