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.

Photo-Excited Dyes: Emerging Technique Against Tau Protein Aggregation.

Tau aggregates are considered a pathological hallmark of Alzheimer's disease. The screening of molecules against Tau aggregation is a novel strategy for Alzheimer's disease. The photo-excited molecules have proven to be effective as a therapeutic agent in several diseases. In recent studies, the photo-excited dyes showed an inhibitory effect on Alzheimer's disease-related Tau protein aggregation and toxicity. The present chapter deals with the effect of rose bengal on the aggregation of Tau. The in vitro studies carried out with the help of electron microscopy, ThS fluorescence, and circular dichroism suggested that RB attenuated the Tau aggregation under in vitro conditions, whereas PE-RB disaggregated the mature Tau fibrils. Photo-excited rose bengal and the classical rose bengal induced a low degree of toxicity in cells. Thus, for the treatment of Alzheimer's disease, the rose bengal could be considered a potential molecule.

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.

Microglia degrade Tau oligomers deposit via purinergic P2Y12-associated podosome and filopodia formation and induce chemotaxis.

BACKGROUND: Tau protein forms neurofibrillary tangles and becomes deposited in the brain during Alzheimer's disease (AD). Tau oligomers are the most reactive species, mediating neurotoxic and inflammatory activity. Microglia are the immune cells in the central nervous system, sense the extracellular Tau via various cell surface receptors. Purinergic P2Y12 receptor can directly interact with Tau oligomers and mediates microglial chemotaxis via actin remodeling. The disease-associated microglia are associated with impaired migration and express a reduced level of P2Y12, but elevate the level of reactive oxygen species and pro-inflammatory cytokines. RESULTS: Here, we studied the formation and organization of various actin microstructures such as-podosome, filopodia and uropod in colocalization with actin nucleator protein Arp2 and scaffold protein TKS5 in Tau-induced microglia by fluorescence microscopy. Further, the relevance of P2Y12 signaling either by activation or blockage was studied in terms of actin structure formations and Tau deposits degradation by N9 microglia. Extracellular Tau oligomers facilitate the microglial migration via Arp2-associated podosome and filopodia formation through the involvement of P2Y12 signaling. Similarly, Tau oligomers induce the TKS5-associated podosome clustering in microglial lamella in a time-dependent manner. Moreover, the P2Y12 was evidenced to localize with F-actin-rich podosome and filopodia during Tau-deposit degradation. The blockage of P2Y12 signaling resulted in decreased microglial migration and Tau-deposit degradation. CONCLUSIONS: The P2Y12 signaling mediate the formation of migratory actin structures like- podosome and filopodia to exhibit chemotaxis and degrade Tau deposit. These beneficial roles of P2Y12 in microglial chemotaxis, actin network remodeling and Tau clearance can be intervened as a therapeutic target in AD.

Photo-Excited Toluidine Blue Disaggregates the Repeat Tau and Modulates End-Binding Protein EB1, Cytoskeletal Structure in Neuronal Cells.

BACKGROUND/AIMS: Alzheimer's disease is a progressive neurological disorder characterized by the intracellular accumulation of Tau protein aggregates. In the present work, we studied the effect of Toluidine Blue and photo-excited Toluidine Blue on the aggregation of repeat Tau using in vitro assays. METHODS: The in vitro experiments were carried out on recombinant repeat Tau which was purified by cation exchange chromatography. The ThS fluorescence analysis was used to study the aggregation kinetics of Tau. CD spectroscopy and electron microscopy were used to study the secondary structure and morphology of Tau respectively. The actin cytoskeleton modulation was studied in Neuro2a cells with help of immunofluorescent microscopy. RESULTS: Results showed that Toluidine Blue efficiently inhibited the formation of higher-order aggregates, which was evidenced by Thioflavin S fluorescence assay, SDS-PAGE, and TEM. Immunofluorescence studies on the cytoskeleton of Neuro2a cells showed that Toluidine Blue and photo-excited Toluidine Blue treatment at a non-toxic concentration of 0.5 µM stimulated the formation of actin-rich lamellipodia and filopodia structures. Tubulin networks were also differentially modulated after the treatment of Toluidine Blue and photo-excited Toluidine Blue. End-binding protein 1 (EB1) levels were observed to increase after Toluidine Blue and photo-excited Toluidine Blue treatment indicating accelerated microtubule polymerization. CONCLUSION: The overall study suggested that Toluidine Blue inhibited the aggregation of soluble Tau and photo-excited Toluidine Blue disaggregated the pre-formed Tau filaments. In our study, TB and PE-TB were observed to be potent against Tau aggregation. We observed a distinctive modulation of actin, tubulin networks, and EB1 levels after TB and PE-TB treatment, which suggested that TB and PE-TB have potency against cytoskeleton deformities.

Tau aggregates improve the Purinergic receptor P2Y12-associated podosome rearrangements in microglial cells.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is associated with protein misfolding, plaque accumulation, neuronal dysfunction, synaptic loss, and cognitive decline. The pathological cascade of AD includes the intracellular Tau hyperphosphorylation and its subsequent aggregation, extracellular Amyloid-ß plaque formation and microglia-mediated neuroinflammation. The extracellular release of aggregated Tau is sensed by surveilling microglia through the involvement of various cell surface receptors. Among all, purinergic P2Y12R signaling is involved in microglial chemotaxis towards the damaged neurons. Microglial migration is highly linked with membrane-associated actin remodeling leading to the phagocytosis of extracellular Tau species. Here, we studied the formation of various actin structures such as podosome, lamellipodia and filopodia, in response to extracellular Tau monomers and aggregates. Microglial podosomes are colocalized with actin nucleator protein WASP, Arp2 and TKS5 adaptor protein during Tau-mediated migration. Moreover, the P2Y12 receptors were associated with F-actin-rich podosome structures, which signify the potential of Tau aggregates in microglial chemotaxis through the involvement of actin remodeling.

TGF-ß1 signalling in Alzheimer's pathology and cytoskeletal reorganization: a specialized Tau perspective.

Microtubule-associated protein, Tau has been implicated in Alzheimer's disease for its detachment from microtubules and formation of insoluble intracellular aggregates within the neurons. Recent findings have suggested the expulsion of Tau seeds in the extracellular domain and their prion-like propagation between neurons. Transforming Growth Factor-ß1 (TGF-ß1) is a ubiquitously occurring cytokine reported to carry out immunomodulation and neuroprotection in the brain. TGF-ß-mediated regulation occurs at the level of neuronal survival and differentiation, glial activation (astrocyte and microglia), amyloid production-distribution-clearance and neurofibrillary tangle formation, all of which contributes to Alzheimer's pathophysiology. Its role in the reorganization of cytoskeletal architecture and remodelling of extracellular matrix to facilitate cellular migration has been well-documented. Microglia are the resident immune sentinels of the brain responsible for surveying the local microenvironment, migrating towards the beacon of pertinent damage and phagocytosing the cellular debris or patho-protein deposits at the site of insult. Channelizing microglia to target extracellular Tau could be a good strategy to combat the prion-like transmission and seeding problem in Alzheimer's disease. The current review focuses on reaffirming the role of TGF-ß1 signalling in Alzheimer's pathology and cytoskeletal reorganization and considers utilizing the approach of TGF-ß-triggered microglia-mediated targeting of extracellular patho-protein, Tau, as a possible potential strategy to combat Alzheimer's disease.

Bacopa monnieri reduces Tau aggregation and Tau-mediated toxicity in cells.

Alzheimer's disease is a neurodegenerative disease characterized by progressive memory loss and behavioral impairments. In the present study, the ethanolic extract of Bacopa monnieri was studied for its potency to inhibit Tau aggregation and rescuing of the viability of Tau-stressed cells. Bacopa monnieri was observed to inhibit the Tau aggregation in vitro. The cells exposed to Bacopa monnieri were also observed to have a low level of ROS and caspase-3 activity. The immunoblot and immunofluorescence analysis showed that Bacopa monnieri acts as an antioxidant and restored the Nrf2 levels in Neuro2a cells. Bacopa monnieri treatment to Neuro2a cells was observed to reduce the phospho-Tau load in formaldehyde-stressed cells. Furthermore, the treatment of Bacopa monnieri reduced the phosphorylation of GSK-3ß in formaldehyde-stressed cells. Ran and NUP358 are the key proteins involved in nuclear transport. It was observed that formaldehyde treatment impaired the nuclear transport by missorting the NUP358 arrangement in Neuro2a cells. On the contrary, Bacopa monnieri treatment restored the NUP358 arrangement in cells. The overall results of the present study suggested that Bacopa monnieri could be considered a potent herb against Tau phosphorylation and Tau aggregation, which projects it as a promising formulation for Alzheimer's disease.

The inhibitory effect of Curcumin-Artemisinin co-amorphous on Tau aggregation and Tau phosphorylation.

Tau is a natively unfolded microtubule-associated protein. Tau neurofibrillary tangles are one of the hallmarks of Alzheimer's disease. The post-translational modifications of Tau lead to its pathological state. Phosphorylation is the key post-translational modification associated with Tauopathy. Curcumin is a polyphenolic compound present in the rhizomes of Curcuma longa. Curcumin has been reported to have remarkable medicinal properties in several diseases, but its poor solubility limits its therapeutic potency. Artemisinin is a sesquiterpene lactone, which has been known sience ancient times for its applications as a treatment for various diseases such as malaria, cancer, autoimmune disease, etc. In the present study, the potency of crystalline curcumin, crystalline artemisinin, and Cur-Art co-amorphous dispersion were evaluated against Tau pathology. The in-vitro ThS/ANS fluorescence and electron microscopy results suggested that curcumin and Cur-Art efficiently inhibited Tau aggregation. Furthermore, exposure to curcumin and Cur-Art co-amorphous restored the impaired nuclear transport in formaldehyde-stressed cells. Curcumin was also found to modulate the phosphorylation of Tau, which indicated the neuroprotective potency. Thus, curcumin and Cur-Art co-amorphous exhibit therapeutic potential against Tau protein in Alzheimer's disease.

Correction: Residue-based propensity of aggregation in the Tau amyloidogenic hexapeptides AcPHF6* and AcPHF6.

[This corrects the article DOI: 10.1039/D0RA03809A.].

Histone deacetylase-6 modulates Tau function in Alzheimer's disease.

Alzheimer's disease (AD), one of the major tauopathies, is multifactorial with a massive demand for disease-modifying treatments rather than symptom management. An AD-affected neuron shows Tau depositions generated due to overload on the proteostasis machinery of the cell and/or abnormal post-translational modifications on Tau protein. Loss of memory or dementia is the most significant concern in AD, occurring due to the loss of neurons and the connections between them. In a healthy brain, neurons interact with the environment and each other through extensions and migratory structures. It can thus be safe to assume that Tau depositions affect these growth structures in neurons. A Histone Deacetylase, HDAC6, has shown elevated levels in AD while also demonstrating direct interaction with the Tau protein. HDAC6 interacts with multiple proteins in the cell and is possibly involved in various signalling pathways. Its deacetylase activity has been a point of controversy in AD; however other functional domains remain unexplored. This review highlights the beneficial potential of HDAC6 in AD in mediating both Tau proteostasis and cytoskeletal rewiring for the neuritic extensions through its Ubiquitin Binding domain (HDAC6 ZnF UBP).

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.

Inter-relationship of Histone Deacetylase-6 with Tau-cytoskeletal organization and remodeling.

Cytoskeletal elements are the key players in cellular integrity, structure, signalling and migration. Each cytoskeletal element comprises of properties with respect to its structure and stability, which serve a specific array of functions. These structures are highly dynamic and regulated by modulation via direct interaction or post-translational modifications. HDAC6 is a cytoplasmic deacetylase known to regulate a wide range of cellular functions either through its deacetylase activity or direct interaction via its C-terminal ZnF UBP domain. HDAC6 has been widely studied for its role in aggresome formation, which acts as a protective mechanism upon protein aggregation. HDAC6 is known to play a critical role in the regulation of cytoskeletal elements-microtubules and actin filaments. This review summarizes the regulatory role of HDAC6 in cytoskeletal remodeling and dynamics of neuronal cells and its significance in neurodegenerative diseases.

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.