Microtubules (MTs) play a fundamental role in many vital processes such as cell division and neuronal activity. They are key structural and functional elements in axons, supporting neurite differentiation and growth, as well as transporting motor proteins along the axons, which use MTs as support tracks. Tau is a stabilizing MT associated protein, whose functions are mainly regulated by phosphorylation. A disruption of the MT network, which might be caused by Tau loss of function, is observed in a group of related diseases called tauopathies, which includes Alzheimer's disease (AD). Tau is found hyperphosphorylated in AD, which might account for its loss of MT stabilizing capacity. Since destabilization of MTs after dissociation of Tau could contribute to toxicity in neurodegenerative diseases, a molecular understanding of this interaction and its regulation is essential.

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Role of Tau as a Microtubule-Associated Protein: Structural and Functional Aspects.

Formation of membrane-less organelles via liquid-liquid phase separation is one way cells meet the biological requirement for spatiotemporal regulation of cellular components and reactions. Recently, tau, a protein known for its involvement in Alzheimer's disease and other tauopathies, was found to undergo liquid-liquid phase separation making it one of several proteins associated with neurodegenerative diseases to do so. Here, we demonstrate that tau forms dynamic liquid droplets in vitro at physiological protein levels upon molecular crowding in buffers that resemble physiological conditions. Tau droplet formation is significantly enhanced by disease-associated modifications, including the AT8 phospho-epitope and the P301L tau mutation linked to an inherited tauopathy. Moreover, tau droplet dynamics are significantly reduced by these modified forms of tau. Extended phase separation promoted a time-dependent adoption of toxic conformations and oligomerization, but not filamentous aggregation. P301L tau protein showed the greatest oligomer formation following extended phase separation. These findings suggest that phase separation of tau may facilitate the formation of non-filamentous pathogenic tau conformations.

/pdf/liquid-liquid-phase-separation-induces-pathogenic-tau-1hymdqxh1e.pdf

Liquid-liquid phase separation induces pathogenic tau conformations in vitro.

Anti-tubulin agents of natural origin: Targeting taxol, vinca, and colchicine binding domains.

BackgroundNeurons are highly polarized cells in which asymmetric axonal-dendritic distribution of proteins is crucial for neuronal function. Loss of polarized distribution of the axonal protein tau is an early sign of Alzheimer’s disease (AD) and other neurodegenerative disorders. The cytoskeletal network in the axon initial segment (AIS) forms a barrier between the axon and the somatodentritic compartment, contributing to axonal retention of tau. Although perturbation of the AIS cytoskeleton has been implicated in neurological disorders, the molecular triggers and functional consequence of AIS perturbation are incompletely understood.ResultsHere we report that tau acetylation and consequent destabilization of the AIS cytoskeleton promote the somatodendritic mislocalization of tau. AIS cytoskeletal proteins, including ankyrin G and βIV-spectrin, were downregulated in AD brains and negatively correlated with an increase in tau acetylated at K274 and K281. AIS proteins were also diminished in transgenic mice expressing tauK274/281Q, a tau mutant that mimics K274 and K281 acetylation. In primary neuronal cultures, the tauK274/281Q mutant caused hyperdynamic microtubules (MTs) in the AIS, shown by live-imaging of MT mobility and fluorescence recovery after photobleaching. Using photoconvertible tau constructs, we found that axonal tauK274/281Q was missorted into the somatodendritic compartment. Stabilizing MTs with epothilone D to restore the cytoskeletal barrier in the AIS prevented tau mislocalization in primary neuronal cultures.ConclusionsTogether, these findings demonstrate that tau acetylation contributes to the pathogenesis of neurodegenerative disease by compromising the cytoskeletal sorting machinery in the AIS.

Additional file 1: Figure S1. of Acetylated tau destabilizes the cytoskeleton in the axon initial segment and is mislocalized to the somatodendritic compartment

Revisiting the intersection of amyloid, pathologically modified tau and iron in Alzheimer's disease from a ferroptosis perspective.

Combretastatin (CA-4) and its analogues are undergoing several clinical trials for treating different types of tumors. In this work, the antiproliferative activity of a series of 2-aminoimidazole-carbonyl analogs of clinically relevant combretastatins A-4 (CA-4) and A-1 was evaluated using a cell-based assay. Among the compounds tested, C-13 and C-21 displayed strong antiproliferative activities against HeLa cells. C-13 inhibited the proliferation of lung carcinoma (A549) cells more potently than combretastatin A-4. C-13 also retarded the migration of A549 cells. Interestingly, C-13 displayed much stronger antiproliferative effects against breast carcinoma and skin melanoma cells compared to noncancerous breast epithelial and skin fibroblast cells. C-13 strongly disassembled cellular microtubules, perturbed the localization of EB1 protein, inhibited mitosis in cultured cells, and bound to tubulin at the colchicine site and inhibited the polymerization of reconstituted microtubules in vitro. C-13 treatment increased the level of reactive oxygen species and induced apoptosis via poly(ADP-ribose) polymerase-cleavage in HeLa cells. The results revealed the importance of the 2-aminoimidazole-carbonyl motif as a double bond replacement in combretastatin and indicated a pharmacodynamically interesting pattern of H-bond acceptors/donors and requisite syn-templated aryls.

https://pubs.acs.org/doi/pdf/10.1021/acsomega.8b00996

Combretastatin-Inspired Heterocycles as Antitubulin Anticancer Agents.

In Alzheimer's disease, tau is predominantly acetylated at K174, K274, K280, and K281 residues. The acetylation of K274-tau is linked with memory loss and dementia. In this study, we have examined the molecular mechanism of the toxicity of acetylated K274-tau. We incorporated an acetylation mimicking mutation at K274 (K→Q) residue of tau. The mutation (K274Q) strongly reduced the ability of tau to bind to tubulin and also to polymerize tubulin while K274R mutation did not reduce the ability of tau either to bind or polymerize tubulin. In addition, K274Q-tau displayed a higher aggregation propensity than wild-type tau as evident from thioflavin S fluorescence, tryptophan fluorescence, and electron microscopic images. Furthermore, dynamic light scattering, atomic force microscopy, and dot blot analysis using an oligomer-specific antibody suggested that K274Q mutation enhanced the oligomerization of tau. The K274Q mutation also strongly decreased the critical concentration for the liquid-liquid phase separation of tau. The oligomeric forms of K274Q-tau were found to be more toxic than wild tau to neuroblastoma cells. Using circular dichroism and fluorescence spectroscopy, we provide evidence indicating that the acetylation mimicking mutation (K274Q) induced conformational changes in tau. The results suggested that the acetylation of tau at 274 residues can increase tau aggregation and enhance the cytotoxicity of tau oligomers.

An acetylation mimicking mutation, K274Q, in tau imparts neurotoxicity by enhancing tau aggregation and inhibiting tubulin polymerization

Photothermal therapy (PTT) is emerging as an effective treatment modality for cancer due to its noninvasive nature. However, the pro-inflammatory necrotic cell death during PTT limits its successfu...

Quercetin Encapsulated Biodegradable Plasmonic Nanoparticles for Photothermal Therapy of Hepatocellular Carcinoma Cells

The aggregation of tau, a microtubule-associated protein, is known to play an important role in several neurological disorders including Alzheimer's disease. Alzheimer's disease is considered to be associated with the dyshomeostasis of metal ions such as aluminum, zinc, copper, and ferric ions. Tau is predominately acetylated at the K274 residue in Alzheimer's disease, and the acetylation of the K274 residue is thought to be linked with dementia. Using acetyl mimicking K274Q mutation in tau, we have examined the effects of the acetylation at K274 residue of tau on the interactions of tau with metal ions and also on the ability of tau to protect DNA from the heat and other stressors. We found that Zn2+ and Al3+ increased the liquid-liquid phase separation of tau, an initial stage of tau aggregation. Further, Zn2+ and Al3+ considerably reduced the critical concentration for the phase separation of K274Q tau. Using far-UV circular dichroism and fluorescence spectroscopy, we provide evidence suggesting that the binding of Zn2+ and Al3+ induces conformational changes in tau. The K274Q mutation enhanced the binding affinity of tau for Zn2+, Al3+, Cu2+, and Fe3+ ions. In addition, Zn2+, Al3+, Cu2+, and Fe3+ significantly enhanced the aggregation propensity of K274Q tau in comparison to tau. Interestingly, tau binds to DNA with a higher affinity than K274Q tau. Tau protects DNA from the DNase treatment in vitro as well as from the heat stress in neuroblastoma cells more efficiently than K274Q tau. The results indicated that the acetylation of K274 residue of tau may increase metal ion-induced toxicity and diminish the ability of tau to protect DNA.

Anuradha Kumari

Papers

Combretastatin-Inspired Heterocycles as Antitubulin Anticancer Agents.

An acetylation mimicking mutation, K274Q, in tau imparts neurotoxicity by enhancing tau aggregation and inhibiting tubulin polymerization

Quercetin Encapsulated Biodegradable Plasmonic Nanoparticles for Photothermal Therapy of Hepatocellular Carcinoma Cells

The Acetyl Mimicking Mutation, K274Q in Tau, Enhances the Metal Binding Affinity of Tau and Reduces the Ability of Tau to Protect DNA.

C12, a combretastatin-A4 analog, exerts anticancer activity by targeting microtubules.