Biology subjects

Chinnathambi, S.

Publications and source records attributed to Chinnathambi, S..

15 recordsLinked to original sources

bioRxiv2026

Neuronal lipid composition regulates distinct states of full-length tau assembly, mechanics and membrane interactions

Aggregation of tau into fibrillar assemblies and neurofibrillary tangles is a defining feature of tauopathies. However, the role of the membrane-rich neuronal environment on the assembly and mechanics of full-length tau remains unclear. Here, we examined how neuronal lipid membranes affect the assembly and membrane interaction of full-length human tau (hTau40). Lipid composition redirected hTau40 assembly, with total brain extract and brain phosphatidylcholine membrane producing shorter, more flexible fibrils, whereas brain phosphatidylserine membrane favoured a more heterogeneous and relatively rigid fibrillar population. Tau-membrane interaction depended strongly on the assembly state of tau. At the same protein concentration, preformed oligomers accumulated on membranes much more rapidly than monomeric tau, indicating that oligomer formation increases membrane recruitment. However, strong membrane binding did not directly predict the extent of membrane perturbation. Pronounced changes in lipid organisation were observed when tau was undergoing continued higher-order assembly at the membrane. Thus, the ability of tau to bind a membrane and its ability to reorganise that membrane appear to be related, but distinct, features of tau-membrane interaction. In cells, internalised hTau40 formed stable, low-mobility assemblies associated with the endolysosomal compartment and changes in its physical properties. These assemblies could also be transferred between cells in a membrane-derived vesicle model. Together, our results show that local membrane composition shapes tau assembly and mechanics, while the state of tau assembly influences how it binds to and remodels membranes. Key words: Amyloid, Tauopathy, Tau protein (Tau), Lipid Vesicle, Membrane Biophysics

biophysics↗

bioRxiv2024

LC3-associated endocytosis facilitates extracellular Tau aggregate internalization and degradation in microglia

BackgroundMicroglia, which are resident phagocytic cells in the brain, are involved in the active clearance of microbes, misfolded proteins, and cell debris, among others. In Alzheimers disease, microglia play a pivotal role in clearing extracellular amyloid-{beta} (A{beta}) plaques and intracellular Tau aggregates from the brain. Microglial cells have several mechanisms for Tau internalization, including macropinocytosis, heparan sulfate proteoglycans (HSPGs), dynamin- dependent endocytosis, and receptor-mediated endocytosis. Internalized Tau seeds either undergo proteasomal or lysosomal degradation or are exocytosed into the extracellular space via exosomes. Microtubule-associated protein 1 light chain 3 (LC3)-associated endocytosis (LANDO) is a recently discovered microglial mechanism for an effective clearance of A{beta} aggregates and alleviates neurodegeneration in murine Alzheimers disease. Several microglial receptors are reported to be involved in misfolded A{beta} and Tau aggregate internalization such as triggering receptor expressed on myeloid cell 2 (TREM-2), P2Y purinoceptor 12 (P2Y12R), C- X3-C motif chemokine receptor 1 (CX3CR1), etc. MethodsIn this study, we report the LANDO of Tau monomers and aggregates by murine microglial cells, which are further degraded by lysosomal fusion. LANDO of extracellular Tau is demonstrated by several biochemical and cell-biology studies including western blotting, fluorescence and confocal imaging of microglial cells. ResultsWe analyzed microglial activation by measuring the upregulation of Ionized Calcium- binding Adapter Molecule 1 (Iba-1) expression. Later, we demonstrated the LANDO of human full-length Tau species, where LC3 colocalizes with internalized Tau, followed by lysosomal degradation by microglia. The accumulation of internalized Tau in the perinuclear region of the cell in the presence of chloroquine supports phagolysosomal fusion and degradation. ConclusionHence, we concluded that microglia internalize extracellular Tau species via LANDO, which further undergoes degradation via the lysosomal pathway.

neuroscience↗

bioRxiv2024

Extracellular HDAC6 ZnF UBP domain enhances podosome-mediated neuronal migration

The increasing prevalence of Alzheimers disease demands research into therapeutic strategies that go beyond Amyloid and Tau. Synaptic loss, neuritic loss, and microtubule destabilization due to misfolded Tau and dysfunctional signalling in the AD-affected neuron, all point toward understanding and targeting cytoskeletal dysregulation in AD. Here we present, an extensive study on the novel role of the ZnF UBP (Zinc Finger Ubiquitin Binding Protein) domain of HDAC6 (Histone Deacetylase 6) in actin remodelling in neurons. We have demonstrated this function through immunofluorescence colocalization analysis in actin-rich podosome structures. We have found that this HDAC6 domain induces increased localization of the actin polymerization proteins, Arp2 and WASP and the adaptor protein TKS5 in the podosome structures. We have also extended our work to understand the potential of this domain in enhancing the podosome-mediated migration of neuronal cells. It was thus established that HDAC6 ZnF UBP induces increased association of cytoskeletal proteins within the podosomes, conferring enhanced migration potential to neurons and presenting an interesting strategy to improve neuronal health.

cell biology↗

bioRxiv2024

Microglia undergoes chemokine receptor, CX3CR1-mediated internalization of extracellular Tau

In Alzheimers disease, the microtubule-associated protein, Tau misfolds to form aggregates and filaments in the intra- and extracellular region of neuronal cells. CX3CR1 receptor is involved in monomeric Tau internalization by directly binding to the receptor. In our study, we are interested in understanding the interaction of different pathological structures of Tau with microglial chemokine receptor, CX3CR1 and its role in CX3CR1-mediated Tau internalization by immunofluorescence assay. Co-immunoprecipitation further confirmed the receptor interaction with Tau oligomers and even aggregated filaments. We next determined the role of Tau as a chemoattractant in regulating the migration of microglia by wound healing assay. Further, immunolocalization studies of CX3CR1 with {beta}-arrestin proved the desensitization of CX3CR1 receptor upon Tau binding. Upon ligand interaction, GPCRs undergoes desensitization and encytosed to the cytosol. We have quantified the CX3CR1 internalization by measuring the membrane to cytosol intensity ratio of CX3CR1 receptor upon extracellular Tau exposure. In addition to experimental assays, a comprehensive analysis of the interaction between the CX3CR1 receptor, the Tau protein, and the AZD8797 ligand has been derived from the combined molecular docking, molecular dynamics and free energy calculations using molecular mechanics-generalized Born approach. In particular, two different molecular dynamics simulations were carried out for CX3CR1-Tau protein and AZD8797-bound CX3CR1-Tau protein interaction. The molecular modeling study establishes that AZD8797 binds to CX3CR1 receptor and modulates the Tau interaction with the receptor.

cell biology↗

bioRxiv2021

G-Protein coupled Purinergic P2Y12 receptor interacts and internalizes TauRD-mediated by membrane-associated actin cytoskeleton remodelling in microglia

In Alzheimers 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 that are involved in constant surveillance and are activated by the extracellular deposits. Purinergic receptors are involved in chemotactic migration of microglial cells towards the site of inflammation. In our recent study, we found that microglial P2Y12 receptor has been involved in phagocytosis of full-length Tau species such as monomers, oligomers and aggregates by actin-driven chemotaxis. In this study, we have showed the interaction of repeat-domain of Tau (TauRD) with microglial P2Y12 receptor and analysed the corresponding residues for interaction by various in-silico approaches. In cellular studies, TauRD was found to interact with microglial P2Y12R and induces its cellular expression as confirmed by coimmunoprecipitation and western blot analysis respectively. Similarly, immunofluorescence microscopic studies emphasized that TauRD were phagocytosed by microglial P2Y12R via the membrane-associated actin remodelling as filopodia extension. Furthermore, the P2Yl2R-mediated TauRD internalization has activated the microglia with an increase in the Iba1 level and TauRD become accumulated at peri-nuclear region as localized with Iba1. Altogether, microglial P2Y12R interacts with TauRD and mediates directed migration and activation for its internalization.

neuroscience↗

bioRxiv2020

EGCG modulates nuclear formaldehyde-induced Tau phosphorylation in Neuronal cells

Tau hyperphosphorylation is one of the major causes of Alzheimers disease pathology. The abnormal phosphorylation curtails the physiological function of Tau of microtubule stabilization and renders it more prone to aggregation. Apart from its function in the cytoplasm, Tau is attributed to play a role in the nucleus. Nuclear function of Tau is dependent on its residue-specific phosphorylation. We studied the effect of a green tea polyphenol, EGCG, on the formaldehyde-induced Tau phosphorylation and Tau kinase CDK5. Interestingly, we observed unique localization of phospho-Tau (AT 8 and AT 100) in the nucleus in various EGCG treatments. EGCG was also found to lower the levels of CDK5 in the formaldehyde-treated cells. Further, the role of EGCG was tested in vivo in drosophila eye model of hyperphosphorylated Tau (Tau E14). The results suggest that EGCG can modulate nuclear Tau phosphorylation and lower the levels of Tau kinase CDK5.

cell biology↗

bioRxiv2020

α- Linoleanic acid modulates phagocytosis of extracellular Tau and induces microglial migration by actin-remodeling

The seeding effect of extracellular Tau species is an emerging aspect to study the Tauopathies in Alzheimers disease. Tau seeds enhance the propagation of disease along with its contribution to microglia-mediated inflammation. Omega-3 fatty acids are known to exert the anti-inflammatory property to microglia by modulating cell membrane compositions that influence various receptors expression and signaling cascade. The immunomodulatory function of omega-3 fatty acids exerts anti-inflammatory properties to microglia. Owing to the imparted anti-inflammatory nature enhance phagocytosis and increased migration property has been observed in microglia. The increased phagocytosis of extracellular Tau monomer and aggregates has been observed upon ALA exposure to microglia cells. The intracellular degradation of internalized Tau species was targeted by early and late endosomal markers Rab5 and Rab7. The increased levels of LAMP-2A and colocalization with internalized Tau indicated the degradation via lysosome. These results indicate the degradation of internalized Tau species in the presence of ALA instead of getting accumulated in the cell. The enhanced migratory ability of microglia in the presence of ALA induces the MTOC repolarization. Tau seeds greatly contribute to the spread of disease, one way to reduce the spreading is to reduce the presence of extracellular Tau seed. Microglia could be influenced to reduce extracellular Tau seed with dietary fatty acids. Our results suggest that dietary fatty acids ALA significantly enhances phagocytosis and intracellular degradation of internalized Tau. Enhanced migration supports the phagocytosis process. Our approach provides insights into the beneficial role of ALA as an anti-inflammatory dietary supplement to treat AD.

neuroscience↗

bioRxiv2020

A green tea polyphenol Epigallocatechin-3-gallate modulates Tau Post-translational modifications and cytoskeletal network

BackgroundAlzheimers disease is a type of dementia denoted by progressive neuronal death due to the accumulation of proteinaceous aggregates of Tau. Post-translational modifications like hyperphosphorylation, truncation, glycation, etc. play a pivotal role in Tau pathogenesis. Glycation of Tau aids in paired helical filament formation and abates its microtubule-binding function. The chemical modulators of Tau PTMs, such as kinase inhibitors and antibody-based therapeutics, have been developed, but natural compounds, as modulators of Tau PTMs are not much explored. MethodsWe applied biophysical and biophysical techniques like fluorescence kinetics, SDS-PAGE, western blot analysis and transmission electron microscopy to investigate the impact of EGCG on Tau glycation in vitro. The effect of glycation on cytoskeleton instability and its EGCG-mediated rescue were studied by immunofluorescence in neuroblastoma cells. ResultsEGCG inhibited methyl glyoxal (MG)-induced Tau glycation in vitro. EGCG potently inhibited MG-induced advanced glycation endproducts formation in neuroblastoma cells as well modulated the localization of AT100 phosphorylated Tau in the cells. In addition to preventing the glycation, EGCG enhanced actin-rich neuritic extensions and rescued actin and tubulin cytoskeleton severely disrupted by MG. EGCG maintained the integrity of the Microtubule Organizing Center (MTOC) stabilized microtubules by Microtubule-associated protein RP/EB family member 1 (EB1). ConclusionsWe report EGCG, a green tea polyphenol, as a modulator of in vitro methylglyoxal-induced Tau glycation and its impact on reducing advanced glycation end products in neuroblastoma cells. We unravel unprecedented function of EGCG in remodeling neuronal cytoskeletal integrity.

cell biology↗

bioRxiv2020

Photo-excited Toluidine Blue disaggregates the Repeat Tau and modulates Cytoskeletal structure in neuronal cells

Alzheimers disease is a progressive neurological disorder characterized by the intracellular accumulation of Tau protein aggregates. Inhibition of protein aggregation by photo-excited dyes is emerging as novel strategy for the treatment of certain diseases. Toluidine Blue is a basic phenothiazine dye having potency of photo-excitation by irradiation with red light at 630{+/-}20 nm. In present work, we studied the effect of Toluidine Blue and photo-excited TB on aggregation of repeat Tau in-vitro using Thioflavin S fluorescence assay, SDS-PAGE and electron microscopy. Results show that TB efficiently inhabited the formation of higher order aggregates. Moreover, the photo-excited TB led to disaggregation of the mature repeat Tau fibrils. Further, studies on the effect of Toluidine blue on cell viability and cytoskeleton network of Neuro2acells show that TB was not toxic to neuronal cells at lower concentrations but at high concentrations (> 5 M) both TB and photo-excited TB induced significant toxicity. Immunofluorescence studies on the cytoskeleton of Neuro2a cells show that Toluidine Blue and photo-excited Toluidine Blue treatment at non-toxic concentration of 0.5 M stimulated 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 the accelerated microtubule polymerization. The overall study suggested that Toluidine Blue inhibited the aggregation of soluble Tau and photo-excited Toluidine Blue disaggregated the pre-formed Tau filaments.

cell biology↗

bioRxiv2019

Melatonin reduces GSK3β expression and tau phosphorylation via Nrf2 nuclear translocation

Alzheimers disease is a neuropathological condition with abnormal formation of extracellular Amyloid-{beta} plaques and intracellular neurofibrillary tangles (NFTs) of Microtubule-associated protein Tau (Tau) in brain. In pathological condition, MAP-Tau can undergo various post-translational modifications such as hyperphosphorylation by the activity of cellular kinases which eventually leads to protein aggregation in neurons. Melatonin is a hormone which mainly secreted from pineal gland, functions to modulate cellular kinases. In our study, we elucidated that Melatonin has inhibited the Tau aggregates mediated cytotoxicity and membrane leakage by MTT and LDH assay respectively in neuro2A cells. Melatonin has found to reduce the GSK3{beta} mRNA expression and protein level by western blot and immunofluorescence assay. Melatonin has also decreased phospho-Tau level (pThr181 and pThr212-pSer214) in neuron cell line upon OA induction as seen by microscopic analysis.. Melatonin treatment has associated with ROS quenching by DCFDA assay, reduced caspase 3 activity in neuronal cells. Further, Melatonin has increased Nrf2 level and nuclear translocation as oxidative stress response in Tauopathy. Together, these findings clearly signifies that Melatonin remediate the Tau-induced neuronal cytotoxicity and reduce Tau hyperphosphorylation via downregulating GSK3{beta} expression. Melatonin can combat oxidative damage by Nrf2 activation and nuclear translocation in AD condition.

cell biology↗

bioRxiv2019

Effect of Melatonin on Tau aggregation and Tau-mediated cell surface morphology

Tau is the major neuronal protein involved in the stabilization of microtubule assembly. In Alzheimers disease, Tau self assembles to form intracellular protein aggregates, which are toxic to cells. Various methods have been tried and tested to restrain the aggregation of Tau. Most of the agents tested for this purpose have limitations in their effectiveness and availability to neuronal cells. We tested melatonin against in vitro Tau aggregation and observed its effect on membrane topology, tubulin network and Tau phosphorylation in neuro2a and N9 cell lines. The aggregation and conformation of Tau was determined by ThT fluorescence and CD spectroscopy respectively. The morphology of Tau aggregates in presence and absence of melatonin was studied by transmission electron microscopy. Melatonin was found to reduce the formation of higher order oligomeric structures without affecting the overall aggregation kinetics of Tau. Melatonin also modulates and helps to maintain membrane topology as evidenced by FE-SEM analysis. Overall, melatonin administration shows mild anti-aggregation and cytoprotective effects.

cell biology↗

bioRxiv2019

Baicalein suppresses Tau fibrillization by sequestering oligomers

Alzheimers disease (AD) is a neurodegenerative disorder caused by protein misfolding, aggregation and accumulation in the brain. A large number of molecules are being screened against these pathogenic proteins but the focus for therapeutics is shifting towards the natural compounds as aggregation inhibitors, mainly due to their minimum adverse effects. Baicalein is a natural compound belonging to the class of flavonoids isolated from the Chinese herb Scutellaria baicalensis. Here we applied fluorescence, absorbance, microscopy, MALDI-TOF spectrophotometry and other biochemical techniques to investigate the interaction between Tau and Baicalein in vitro. We found the aggregation inhibitory properties of Baicalein for the repeat Tau. Overall, the potential of Baicalein in dissolving the preformed Tau oligomers as well as mature fibrils can be of utmost importance in therapeutics for Alzheimers disease.

biochemistry↗

bioRxiv2019

Modulation of Actin network and Tau phosphorylation by HDAC6 ZnF UBP domain

Microtubule-associated protein Tau undergoes aggregation in Alzheimers disease and a group of other related diseases collectively known as Tauopathies. In AD, Tau forms aggregates, which are deposited intracellularly as neurofibrillary tangles. HDAC6 plays an important role in aggresome formation where it recruits polyubiquitinated aggregates to the motor protein dynein. Here, we have studied the effect of HDAC6 ZnF UBP on Tau phosphorylation, ApoE localization, GSK-3{beta} regulation and cytoskeletal organization in neuronal cells by immunocytochemistry. Immunocytochemistry reveals that HDAC6 ZnF UBP can modulate Tau phosphorylation and actin cytoskeleton organization when the cells are exposed to the domain. HDAC6 ZnF UBP treatment to cells does not affect their viability and resulted in enhanced neurite extension and formation of structures similar to podosomes, lamellipodia and podonuts suggesting its role in actin re-organization. Also, HDAC6 treatment showed increased nuclear localization of ApoE and tubulin localization in microtubule organizing centre. Our studies suggest the regulatory role of this domain in different aspects of neurodegenerative diseases.

cell biology↗

bioRxiv2018

Molecular Complexes for Effective Inhibition of Tau Aggregation

Tau is an axonal protein known to form abnormal aggregates and is the biomarker of Alzheimers disease. Metal-based therapeutics for inhibition of Tau aggregation is limited and rarely reported in the contemporary science. Here, the first example is reported of a rationally designed molecular cobalt(II)-complexes for effective inhibition of Tau and disaggregation of preformed Tau fibrils. The mechanistic studies revealed that the prevention of Tau aggregation by CBMCs is concentration-dependent and Tau seldom exhibits conformational changes. Interestingly, CBMCs play a dual role by causing disassembly of preformed aggregates as well as complete Tau inhibition. We believe that this unprecedented finding by the newly developed molecular complexes has a potential to lead to developing innovative metal-based therapeutics for Alzheimers disease.

neuroscience↗

bioRxiv2018

Substrate deformations induce directed keratinocyte migration

Cell migration is an essential part of many (patho)physiological processes in the body, including keratinocyte re-epithelialization of healing wounds. Recent interest in the mechanobiology of tissues suggests that physical forces and mechanical cues from the wound bed (in addition to biochemical signals) may also play an important role in the healing process. Previously, we explored this possibility and found that polyacrylamide (PA) gel stiffness affected primary human keratinocyte behavior and that mechanical deformations in soft (~1.2 kPA) PA gels produced by neighboring cells appeared to influence the process of de novo epithelial sheet formation. In order to clearly demonstrate that keratinocytes do respond to such deformations, we conducted a series of experiments where we observed the response of single keratinocytes to a prescribed local substrate deformation that mimicked a neighboring cell or evolving multicellular aggregate via a servo-controlled microneedle. We also examined the effect of adding either Y27632, a rho kinase inhibitor, or blebbistatin, a non-muscle myosin II inhibitor, on the response of the cells to PA gel deformations. The results of this study indicate that keratinocytes do sense and respond to mechanical signals comparable to those that originate from substrate displacements imposed by neighboring cells, a finding that could have important implications for the process of keratinocyte re-epithelialization that takes place during normal and pathologic wound healing. Furthermore, the Rho/ROCK pathway and the engagement of NM II are both essential to the observed process of substrate deformation-directed keratinocyte migration.

biophysics↗