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Metabolic reactions within cells occur in various isolated compartments with or without borders, the latter being known as membrane-less organelles (MLOs). The MLOs show liquid-like properties and are formed by a process known as liquid-liquid phase separation (LLPS). MLOs contribute to different molecules interactions such as protein-protein, protein-RNA, and RNA-RNA driven by various factors, such as multivalency of intrinsic disorders. MLOs are involved in several cell signaling pathways such as transcription, immune response, and cellular organization. However, disruption of these processes has been found in different pathologies. Recently, it has been demonstrated that protein aggregates, a characteristic of some neurodegenerative diseases, undergo similar phase separation. Tau protein is known as a major neurofibrillary tangles component in Alzheimer's disease (AD). This protein can undergo phase separation to form a MLO known as tau droplet in vitro and in vivo, and this process can be facilitated by several factors, including crowding agents, RNA, and phosphorylation. Tau droplet has been shown to mature into insoluble aggregates suggesting that this process may precede and induce neurodegeneration in AD. Here we review major factors involved in liquid droplet formation within a cell. Additionally, we highlight recent findings concerning tau aggregation following phase separation in AD, along with the potential therapeutic strategies that could be explored in this process against the progression of this pathology.

Ten macrocyclic peptides, each comprising 14 amino acids, were designed and synthesized based on the Tau aggregation model hexapeptides AcPHF6* and AcPHF6. The design took into account the aggregation tendencies of each residue in AcPHF6* and AcPHF6, their aggregation models, while employing peptide-based structural design principles including N-methylation to promote turns and to block hydrogen bond propagation and elongation of the aggregation chain. NMR analysis supported that all these peptides adopted an antiparallel β-sheet conformation. Self-aggregation studies characterized the aggregation properties of these peptides, identifying two peptides with the highest (P3) and lowest (P8) aggregation tendencies. In cross-aggregation studies with the parent peptides AcPHF6* and AcPHF6, P3 and P8 were found to promote and reduce aggregation, respectively. Furthermore, P3 and P8 demonstrated an enhancement and diminution effect on the aggregation of K18wt, indicating their capacity to modulate aggregation even at the macromolecular level. Thus, the two simple peptides, P3 and P8 selectively exhibit pro- or anti-aggregation effects on PHF peptides and Tau. This study, has thus developed structurally well-defined non-complex peptides, derived from AcPHF6* and AcPHF6, to modulate Tau aggregation as desired, offering applications in Tau model studies and the development of Tau aggregation inhibitors or promoters., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The accumulation of hyperphosphorylated fibrillar Tau aggregates in the brain is one of the defining hallmarks of Tauopathy diseases, including Alzheimer's disease. However, the primary events or molecules responsible for initiation of the pathological Tau aggregation and spreading remain unknown. The discovery of heparin as an effective inducer of Tau aggregation in vitro was instrumental to enabling different lines of research into the role of Tau aggregation in the pathogenesis of Tauopathies. However, recent proteomics and cryogenic electron microscopy (cryo-EM) studies have revealed that heparin-induced Tau fibrils generated in vitro do not reproduce the biochemical and ultrastructural properties of disease-associated brain-derived Tau fibrils. These observations demand that we reassess our current approaches for investigating the mechanisms underpinning Tau aggregation and pathology formation. Our review article presents an up-to-date survey and analyses of 1) the evolution of our understanding of the interactions between Tau and heparin, 2) the various structural and mechanistic models of the heparin-induced Tau aggregation, 3) the similarities and differences between brain-derived and heparin-induced Tau fibrils; and 4) emerging concepts on the biochemical and structural determinants underpinning Tau pathological heterogeneity in Tauopathies. Our analyses identify specific knowledge gaps and call for 1) embracing the complexities of Tau pathologies; 2) reassessment of current approaches to investigate, model and reproduce pathological Tau aggregation as it occurs in the brain; 3) more research towards a better understanding of the naturally-occurring cofactor molecules that are associated with Tau brain pathology initiation and propagation; and 4) developing improved approaches for in vitro production of the Tau aggregates and fibrils that recapitulate and/or amplify the biochemical and structural complexity and diversity of pathological Tau in Tauopathies. This will result in better and more relevant tools, assays, and mechanistic models, which could significantly improve translational research and the development of drugs and antibodies that have higher chances for success in the clinic.

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-3β 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., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest with the contents of this article., (Copyright © 2023. Published by Elsevier B.V.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Disease-related tau protein in Alzheimer's disease is hyperphosphorylated and aggregates into neurofibrillary tangles. The cis-proline isomer of the pSer/Thr-Pro sequence has been proposed to act as a precursor of aggregation ('Cistauosis' hypothesis), but this aggregation scheme is not yet entirely accepted. Hence to investigate isomer-specific-aggregation of tau, proline residues at the RTPPK motif were replaced by alanine residues (with permanent trans configuration) employing genetic engineering methods. RTPAK, RTAPK, and RTAAK mutant variants of tau were generated, and their in vitro aggregation propensity was investigated using multi-spectroscopic techniques. Besides, the cell toxicity of oligomers/fibrils was analyzed and compared to those of the wild-type (WT) tau. Analyses of mutant variants have shown to be in agreement (to some degree) to the theory of the 'cistauosis' hypothesis. The results showed that the trans isomer in the 232-rd residue (P232A mutant rather than P233A) had reduced aggregation propensity. However, this study did not illustrate any statistically significant difference between the wild and the mutant protein aggregations concerning cell toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no financial interests or personal relationships that could have appeared to influence the work reported in the present study., (Copyright © 2022. Published by Elsevier B.V.)

Development of multitargeted ligands have demonstrated remarkable efficiency as potential therapeutics for Alzheimer's disease (AD). Herein, we reported a new series of deoxyvasicinone analogues as dual inhibitor of acetylcholinesterase (AChE) and tau aggregation that function as multitargeted ligands for AD. All the multitargeted ligands 11(a-j) and 15(a-g) were designed, synthesized, and validated by 1 HNMR, 13 CNMR and mass spectrometry. All the synthesized compounds 11(a-j) and 15(a-g) were screened for their ability to inhibit AChE, BACE1, amyloid fibrillation, α-syn aggregation, and tau aggregation. All the screened compounds possessed weak inhibition of BACE-1, Aβ 42 and α-syn aggregation. However, several compounds were identified as potential hits in the AChE inhibitory screening assay and cellular tau aggregation screening. Among all compounds, 11f remarkably inhibited AChE activity and cellular tau oligomerization at single-dose screening (10 µM). Moreover, 11f displayed a half-maximal inhibitory concentration (IC 50 ) value of 0.91 ± 0.05 µM and half-maximal effective concentration (EC 50 ) value of 3.83 ± 0.51 µM for the inhibition of AChE and cellular tau oligomerization, respectively. In addition, the neuroprotective effect of 11f was determined in tau-expressing SH-SY5Y cells incubated with Aβ oligomers. These findings highlighted the potential of 11f to function as a multifunctional ligand for the development of promising anti-AD drugs., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Neuronal hyperexcitability linked to an increase in glutamate signalling is a peculiar trait of the early stages of Alzheimer's disease (AD) and tauopathies, however, a progressive reduction in glutamate release follows in advanced stages. We recently reported that in the early phases of the neurodegenerative process, soluble, non-aggregated Tau accumulates in the nucleus and modulates the expression of disease-relevant genes directly involved in glutamatergic transmission, thus establishing a link between Tau instability and altered neurotransmission. Here we report that while the nuclear translocation of Tau in cultured cells is not impaired by its own aggregation, the nuclear amyloid inclusions of aggregated Tau abolish Tau-dependent increased expression of the glutamate transporter. Remarkably, we observed that in the prefrontal cortex (PFC) of AD patient brain, the glutamate transporter is upregulated at early stages and is downregulated at late stages. The Gene Set Enrichment Analysis indicates that the modulation of Tau-dependent gene expression along the disease progression can be extended to all protein pathways of the glutamatergic synapse. Together, this evidence links the altered glutamatergic function in the PFC during AD progression to the newly discovered function of nuclear Tau., Competing Interests: Declaration of Interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Tauopathy belongs to a various class of neurodegenerative diseases in which insoluble Tau aggregates are formed, and cellular function is lost, leading to neuronal death. Various therapeutic strategies have been investigated, and many drugs have been proposed as a cure for these diseases but their toxicity and adverse side effects, limit their consumption by humans. Alternatively, the use of non-toxic medicine without any adverse or undesirable secondary effects like nutrients, vitamins, as well as herbal and mineral supplements is common and continues to increase each year. Folic acid is a form of vitamin B and plays a vital role in the synthesis of nucleic acid, methionine regeneration, and in shuttling one-carbon units essential for normal cell division and growth. We investigated the interaction between folic acid and Tau protein, then experimental and theoretical evidence were provided for the suppressing effects of folic acid on Tau fibril formation. The obtained results showed that folic acid could interact with Tau through a spontaneous binding process, mainly by hydrophobic forces, and this interaction leads to a decline in protein-protein interactions through stabilizing its native state, limiting successful Tau-seed oligomerization and consequently decelerating polymerization of Tau amyloid aggregates., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as potential conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a fatal neurodegenerative disorder with an alarming increase in the death rate every year. AD is characterised by an aberrant accumulation of proteins in the form of aggregates. The axonal microtubule-associated protein Tau and amyloid-β undergo structural transition to β-sheet rich structure and form aggregates in neuronal soma as well as in the extracellular region. The loss of Tau from microtubules leads to the disintegration of axon and causing neuronal degeneration. This led to the development of effective drugs against AD, to prevent Tau aggregation. Here, we synthesized and screen metal-based complexes to prevent Tau protein aggregation. ThS fluorescence and TEM suggested the role of synthetic cobalt complexes in inhibiting Tau aggregation. CD spectroscopy showed that these complexes prevented conformational changes in Tau to β-sheet. CBMCs were not toxic at lower concentrations and formed non-toxic Tau species. L1 and L2 prevented membrane leakage; whereas, higher concentrations of L3 caused membrane leakage as observed by LDH release assay. The overall results indicate the synthetic cobalt complexes to be a promising molecule against AD., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Aggregation of tau into paired helical filament (PHF) is a hallmark of Alzheimer's disease (AD), and Cys-mediated disulfide bond formation plays a vital role in tau fibrillation. While intermolecular disulfide bond between Cys residues in microtubule-binding repeat (MTBR) region facilitates tau aggregation, intramolecular disulfide bond attenuates the same, though the molecular basis for such phenomenon remains obscure. Thus intramolecular disulfide-bonded tau monomer might be an excellent model to understand the unique features of aggregation-resistant tau conformer., Methods: We synthesized the Cys cross-linked tau40 monomer by oxidation and characterized the altered conformational dynamics in the molecule by Hydrogen-deuterium exchange, limited proteolysis and fluorescence quenching., Results: Deuterium exchange study showed that rigidity was imparted in the core PHF region of oxidized tau40 in MTBR segment, consisting of the fundamental PHF6 motif. Conformational rigidity was prominent in C-terminal tail region also. Limited proteolysis supported reduced accessibility of MTBR region in the molecule., Conclusions: PHF formation of oxidized tau40 might be attenuated either by induction of intramolecular H-bonding between the regions of high β-structure propensity in second and third MTBR (R 2 , R 3 ), thus preventing intermolecular interaction between them, or by imparted rigidity in R 2 -R 3 , preventing the formation of extended β-structure preceding fibrillation. Data indicated plausible effect of conformational adaptation on the nucleation process of oxidized tau40 assembly., General Significance: Our findings unravel the essential molecular features of aggregation-resistant tau conformer. Therapeutics stabilizing such conformers in vivo might be of high benefit in arresting tau assembly during AD and other tauopathies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We have developed a cell-based phenotypic automated high-content screening approach for N2a cells expressing the pro-aggregant repeat domain of tau protein (tau RDΔK ), which allows analysis of a chemogenomic library of 1649 compounds for their effect on the inhibition or stimulation of intracellular tau aggregation. We identified several inhibitors and stimulators of aggregation and achieved a screening reproducibility >85% for all data. We identified 18 potential inhibitors (= 1.1% of the library) and 10 stimulators (= 0.6% of the library) of tau aggregation in this cell model of tau pathology. The results provide insights into the regulation of cellular tau aggregation and the pathways involved in this process (e.g., involving signaling via p38 mitogen-activated protein kinase, histone deacetylases, vascular endothelial growth factor, rho/ROCK). For example, inhibitors of protein kinases (e.g., p38) can reduce tau aggregation, whereas inhibitors of deacetylases (histone deacetylases) can enhance aggregation. These observations are compatible with reports that phosphorylated or acetylated tau promotes pathology., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Alzheimer disease neuropathology is characterized by the extracellular accumulation of Aβ peptide and intracellular aggregation of hyperphosphorylated tau. With the progression of the disease, macroscopic atrophy affects the entorhinal area and hippocampus, amygdala, and associative regions of the neocortex. The locus coeruleus is depigmented. The deposition of Aβ is first made of diffuse deposits. Amyloid focal deposits constitute the core of the senile plaque which also comprises a corona of tau-positive neurites. Aβ deposits are found successively in the neocortex, the hippocampus, the striatum, the mesencephalon, and finally the cerebellum together with the pontine nuclei (Thal phases). Tau pathology affects in a stereotyped order some specific nuclei of the brainstem, the entorhinal area, the hippocampus, and the neocortex - first the associative areas and secondarily the primary cortices (Braak stages). Loss of synapses is observed in association with tau and Aβ pathology; neuronal loss occurs in the most affected areas. Granulovacuolar degeneration and perisomatic granules are also linked to Alzheimer disease pathology. The physiopathology of Alzheimer disease remains unknown. Familial cases suggest that Aβ deposition is the initial step, but tau pathology appears early in the course and seems to be better correlated with the symptoms., (Copyright © 2017 Elsevier B.V. All rights reserved.)