Fluorescent Phenotyping of Blood Cells Using a Differential Sensing Strategy: Differentiating Physiological Aging Stages and Neuro-Degenerative Disease Drugs.

Blood continually contributes to the maintenance of homeostasis of the body and contains information regarding the health state of an individual. However, current hematological analyses predominantly rely on a limited number of CD markers and morphological analysis. In this work, differentially sensitive fluorescent compounds based on TCF scaffolds are introduced that are designed for fluorescent phenotyping of blood. Depending on their structures, TCF compounds displayed varied responses to reactive oxygen species, biothiols, redox-related biomolecules, and hemoglobin, which are the primary influential factors within blood. Contrary to conventional CD marker-based analysis, this unbiased fluorescent phenotyping method produces diverse fingerprints of the health state. Precise discrimination of blood samples from 37 mice was demonstrated based on their developmental stages, ranging from 10 to 19 weeks of age. Additionally, this fluorescent phenotyping method enabled the differentiation between drugs with distinct targets, serving as a simple yet potent tool for pharmacological analysis to understand the mode of action of various drugs.

Structural determinants for activation of the Tau kinase CDK5 by the serotonin receptor 5-HT7R.

BACKGROUND: Multiple neurodegenerative diseases are induced by the formation and deposition of protein aggregates. In particular, the microtubule-associated protein Tau leads to the development of so-called tauopathies characterized by the aggregation of hyperphosphorylated Tau within neurons. We recently showed that the constitutive activity of the serotonin receptor 7 (5-HT7R) is required for Tau hyperphosphorylation and aggregation through activation of the cyclin-dependent kinase 5 (CDK5). We also demonstrated physical interaction between 5-HT7R and CDK5 at the plasma membrane suggesting that the 5-HT7R/CDK5 complex is an integral part of the signaling network involved in Tau-mediated pathology. METHODS: Using biochemical, microscopic, molecular biological, computational and AI-based approaches, we investigated structural requirements for the formation of 5-HT7R/CDK5 complex. RESULTS: We demonstrated that 5-HT7R domains responsible for coupling to Gs proteins are not involved in receptor interaction with CDK5. We also created a structural model of the 5-HT7R/CDK5 complex and refined the interaction interface. The model predicted two conserved phenylalanine residues, F278 and F281, within the third intracellular loop of 5-HT7R to be potentially important for complex formation. While site-directed mutagenesis of these residues did not influence Gs protein-mediated receptor signaling, replacement of both phenylalanines by alanine residues significantly reduced 5-HT7R/CDK5 interaction and receptor-mediated CDK5 activation, leading to reduced Tau hyperphosphorylation and aggregation. Molecular dynamics simulations of 5-HT7R/CDK5 complex for wild-type and receptor mutants confirmed binding interface stability of the initial model. CONCLUSIONS: Our results provide a structural basis for the development of novel drugs targeting the 5-HT7R/CDK5 interaction interface for the selective treatment of Tau-related disorders, including frontotemporal dementia and Alzheimer's disease.

NeuM: A Neuron-Selective Probe Incorporates into Live Neuronal Membranes via Enhanced Clathrin-Mediated Endocytosis in Primary Neurons.

The development of a small-molecule probe designed to selectively target neurons would enhance the exploration of intricate neuronal structures and functions. Among such probes, NeuO stands out as the pioneer and has gained significant traction in the field of research. Nevertheless, neither the mechanism behind neuron-selectivity nor the cellular localization has been determined. Here, we introduce NeuM, a derivative of NeuO, designed to target neuronal cell membranes. Furthermore, we elucidate the mechanism behind the selective neuronal membrane trafficking that distinguishes neurons. In an aqueous buffer, NeuM autonomously assembles into micellar structures, leading to the quenching of its fluorescence (Φ=0.001). Upon exposure to neurons, NeuM micelles were selectively internalized into neuronal endosomes via clathrin-mediated endocytosis. Through the endocytic recycling pathway, NeuM micelles integrate into neuronal membrane, dispersing fluorescent NeuM molecules in the membrane (Φ=0.61). Molecular dynamics simulations demonstrated that NeuM, in comparison to NeuO, possesses optimal lipophilicity and molecular length, facilitating its stable incorporation into phospholipid layers. The stable integration of NeuM within neuronal membrane allows the prolonged monitoring of neurons, as well as the visualization of intricate neuronal structures.

Synthesis and biological evaluation of indane-based fluorescent probes for detection of amyloid-ß aggregates in Alzheimer's disease.

In this article, the development of fluorescent imaging probes for the detection of Alzheimer's disease (AD)-associated protein aggregates is described. Indane derivatives with a donor-π-acceptor (D-π-A) structure were designed and synthesized. The probes were evaluated for their ability to bind to ß-amyloid (Aß) protein aggregates, which are a key pathological hallmark of AD. The results showed that several probes exhibited significant changes in fluorescence intensity at wavelengths greater than 600 nm when they were bound to Aß aggregates compared to the Aß monomeric form. Among the tested probes, four D-π-A type indane derivatives showed promising binding selectivity to Aß aggregates over non-specific proteins such as bovine serum albumin (BSA). The molecular docking study showed that our compounds were appropriately located along the Aß fibril axis through the hydrophobic tunnel structure. Further analysis revealed that the most active compound having dimethylaminopyridyl group as an election donor and dicyano group as an electron acceptor could effectively stain Aß plaques in brain tissue samples from AD transgenic mice. These findings suggest that our indane-based compounds have the potential to serve as fluorescent probes for the detection and monitoring of Aß aggregation in AD.

Amisulpride as a potential disease-modifying drug in the treatment of tauopathies.

INTRODUCTION: Hyperphosphorylation and aggregation of the microtubule-associated protein tau cause the development of tauopathies, such as Alzheimer's disease and frontotemporal dementia (FTD). We recently uncovered a causal link between constitutive serotonin receptor 7 (5-HT7R) activity and pathological tau aggregation. Here, we evaluated 5-HT7R inverse agonists as novel drugs in the treatment of tauopathies. METHODS: Based on structural homology, we screened multiple approved drugs for their inverse agonism toward 5-HT7R. Therapeutic potential was validated using biochemical, pharmacological, microscopic, and behavioral approaches in different cellular models including tau aggregation cell line HEK293 tau bimolecular fluorescence complementation, primary mouse neurons, and human induced pluripotent stem cell-derived neurons carrying an FTD-associated tau mutation as well as in two mouse models of tauopathy. RESULTS: Antipsychotic drug amisulpride is a potent 5-HT7R inverse agonist. Amisulpride ameliorated tau hyperphosphorylation and aggregation in vitro. It further reduced tau pathology and abrogated memory impairment in mice. DISCUSSION: Amisulpride may be a disease-modifying drug for tauopathies.

Cereblon Regulates the Proteotoxicity of Tau by Tuning the Chaperone Activity of DNAJA1.

Protein aggregation can induce explicit neurotoxic events that trigger a number of presently untreatable neurodegenerative disorders. Chaperones, on the other hand, play a neuroprotective role because of their ability to unfold and refold abnormal proteins. The progressive nature of neurotoxic events makes it important to discover endogenous factors that affect pathologic and molecular phenotypes of neurodegeneration in animal models. Here, we identified microtubule-associated protein tau, and chaperones Hsp70 (heat shock protein 70) and DNAJA1 (DJ2) as endogenous substrates of cereblon (CRBN), a substrate-recruiting subunit of cullin4-RING-E3-ligase. This recruitment results in ubiquitin-mediated degradation of tau, Hsp70, and DJ2. Knocking out CRBN enhances the chaperone activity of DJ2, resulting in decreased phosphorylation and aggregation of tau, improved association of tau with microtubules, and reduced accumulation of pathologic tau across brain. Functionally abundant DJ2 could prevent tau aggregation induced by various factors like okadaic acid and heparin. Depletion of CRBN also decreases the activity of tau-kinases including GSK3α/ß, ERK, and p38. Intriguingly, we found a high expression of CRBN and low levels of DJ2 in neuronal tissues of 5XFAD and APP knock-in male mouse models of Alzheimer's disease. This implies that CRBN-mediated DJ2/Hsp70 pathway may be compromised in neurodegeneration. Being one of the primary pathogenic events, elevated CRBN can be a contributing factor for tauopathies. Our data provide a functional link between CRBN and DJ2/Hsp70 chaperone machinery in abolishing the cytotoxicity of aggregation-prone tau and suggest that Crbn-/- mice serve as an animal model of resistance against tauopathies for further exploration of the molecular mechanisms of neurodegeneration.

Contagious Aggregation: Transmittable Protein Aggregation in Cellular Communities Initiated by Synthetic Cells.

Aggregation of amyloidogenic proteins causing neurodegenerative diseases is an uncontrollable and contagious process that is often associated with lipid membranes in a highly complex physiological environment. Although several approaches using natural cells and membrane models have been reported, systematic investigations focusing on the association with the membranes are highly challenging, mostly because of the lack of proper molecular tools. Here, we report a new supramolecular approach using a synthetic cell system capable of controlling the initiation of protein aggregation and mimicking various conditions of lipid membranes, thereby enabling systematic investigations of membrane-dependent effects on protein aggregation by visualization. Extending this strategy through concurrent use of synthetic cells and natural cells, we demonstrate the potential of this approach for systematic and in-depth studies on interrogating inter- and intracellularly transmittable protein aggregation. Thus, this new approach offers opportunities for gaining insights into the pathological implications of contagious protein aggregation associated with membranes for neurotoxicity.

Modulation of SETDB1 activity by APQ ameliorates heterochromatin condensation, motor function, and neuropathology in a Huntington's disease mouse model.

The present study describes evaluation of epigenetic regulation by a small molecule as the therapeutic potential for treatment of Huntington's disease (HD). We identified 5-allyloxy-2-(pyrrolidin-1-yl)quinoline (APQ) as a novel SETDB1/ESET inhibitor using a combined in silico and in vitro cell based screening system. APQ reduced SETDB1 activity and H3K9me3 levels in a HD cell line model. In particular, not only APQ reduced H3K9me3 levels in the striatum but it also improved motor function and neuropathological symptoms such as neuronal size and activity in HD transgenic (YAC128) mice with minimal toxicity. Using H3K9me3-ChIP and genome-wide sequencing, we also confirmed that APQ modulates H3K9me3-landscaped epigenomes in YAC128 mice. These data provide that APQ, a novel small molecule SETDB1 inhibitor, coordinates H3K9me-dependent heterochromatin remodelling and can be an epigenetic drug for treating HD, leading with hope in clinical trials of HD.

Pan-HDAC Inhibitors Promote Tau Aggregation by Increasing the Level of Acetylated Tau.

Epigenetic remodeling via histone acetylation has become a popular therapeutic strategy to treat Alzheimer's disease (AD). In particular, histone deacetylase (HDAC) inhibitors including M344 and SAHA have been elucidated to be new drug candidates for AD, improving cognitive abilities impaired in AD mouse models. Although emerged as a promising target for AD, most of the HDAC inhibitors are poorly selective and could cause unwanted side effects. Here we show that tau is one of the cytosolic substrates of HDAC and the treatment of HDAC inhibitors such as Scriptaid, M344, BML281, and SAHA could increase the level of acetylated tau, resulting in the activation of tau pathology.

Metal Ion Effects on Aß and Tau Aggregation.

Amyloid and tau aggregation are implicated in manifold neurodegenerative diseases and serve as two signature pathological hallmarks in Alzheimer's disease (AD). Though aging is considered as a prominent risk factor for AD pathogenesis, substantial evidence suggests that an imbalance of essential biometal ions in the body and exposure to certain metal ions in the environment can potentially induce alterations to AD pathology. Despite their physiological importance in various intracellular processes, biometal ions, when present in excessive or deficient amounts, can serve as a mediating factor for neurotoxicity. Recent studies have also demonstrated the contribution of metal ions found in the environment on mediating AD pathogenesis. In this regard, the neuropathological features associated with biometal ion dyshomeostasis and environmental metal ion exposure have prompted widespread interest by multiple research groups. In this review, we discuss and elaborate on findings from previous studies detailing the possible role of both endogenous and exogenous metal ions specifically on amyloid and tau pathology in AD.

Visualization of Tau⁻Tubulin Interaction in a Living Cell Using Bifluorescence Complementation Technique.

Tau is a neuron-specific microtubule-binding protein that stabilizes microtubules. It is generally thought that highly phosphorylated tau dissociates from microtubules and becomes insoluble aggregates, leading to neuronal degeneration. Due to the implication of tau aggregation in neurodegenerative disorders, including Alzheimer's disease, great efforts have been made to identify the tau aggregation process. However, tau interaction with tubulin during the aggregation process remains largely unknown. To scrutinize the tau-tubulin interaction, we generated a cell model that enables visualization of the tau-tubulin interaction in a living cell using the Bifluorescence Complementation (BiFC) Technique. Upon diverse chemical stimulation that induced tau pathology, tau-tubulin BiFC cells showed significantly increased levels of BiFC fluorescence, indicating that tau aggregates together with tubulin. Our results suggest that tubulin should be considered as a key component in the tau aggregation process.

Monitoring of Intracellular Tau Aggregation Regulated by OGA/OGT Inhibitors.

Abnormal phosphorylation of tau has been considered as a key pathogenic mechanism inducing tau aggregation in multiple neurodegenerative disorders, collectively called tauopathies. Recent evidence showed that tau phosphorylation sites are protected with O-linked ß-N-acetylglucosamine (O-GlcNAc) in normal brain. In pathological condition, tau is de-glycosylated and becomes a substrate for kinases. Despite the importance of O-GlcNAcylation in tau pathology, O-GlcNAc transferase (OGT), and an enzyme catalyzing O-GlcNAc to tau, has not been carefully investigated in the context of tau aggregation. Here, we investigated intracellular tau aggregation regulated by BZX2, an inhibitor of OGT. Upon the inhibition of OGT, tau phosphorylation increased 2.0-fold at Ser199 and 1.5-fold at Ser396, resulting in increased tau aggregation. Moreover, the BZX2 induced tau aggregation was efficiently reduced by the treatment of Thiamet G, an inhibitor of O-GlcNAcase (OGA). Our results demonstrated the protective role of OGT in tau aggregation and also suggest the counter-regulatory mechanism of OGA and OGT in tau pathology.

User tendency-based rating scaling in online trading networks.

Social networks often involve the users rating each other based on their beliefs, abilities, and other characteristics. This is particularly common in e-commerce platforms where buyers rate sellers based on their trustworthiness. However, the rating tends to vary between users due to differences in their individual scoring criteria. For example, in a transaction network, a positive user may give a high rating unless the transaction was unsatisfactory while a neutral user may give a mid-rating, consequently giving the same numeric score to different levels of satisfaction. In this paper, we propose a novel method called user tendency-based rating scaling, which adjusts the current rating (its score) based on the pattern of past ratings. We investigate whether this rating scaling method can classify between "good users" and "bad users" in online trade social networks better when compared with using the original rating scores without scaling. Classifying between good users and bad users is especially important for anonymous rating networks like Bitcoin transaction networks, where users' reputations must be recorded to preclude fraudulent and risky users. We evaluate the proposed rating scaling method by performing user classification, link prediction, and clustering tasks, using three real-world online rating network datasets. We use both the original ratings and the scaled ratings as weights of graphs and use a weighted graph embedding method to find node representations that reflect users' positive and negative information. The experimental results showed that using the proposed rating scaling method outperformed using the original (i.e., unscaled) ratings by up to 17% in classification accuracy, and by up to 2.5% in link prediction based on the AUC ROC measure, and by up to 21% in the clustering tasks based on the Dunn-index.

An amphiphilic material arginine-arginine-bile acid promotes α-synuclein amyloid formation.

Amyloid generation plays essential roles in various human diseases, biological functions, and nanotechnology. However, developing efficient chemical and biological candidates for regulating amyloid fibrillation remains difficult because information on the molecular actions of modulators is insufficient. Thus, studies are needed to understand how the intermolecular physicochemical properties of the synthesised molecules and amyloid precursors influence amyloidogenesis. In this study, we synthesised a novel amphiphilic sub-nanosized material, arginine-arginine (RR)-bile acid (BA), by conjugating positively charged RR to hydrophobic BA. The effects of RR-BA on amyloid formation were investigated on α-synuclein (αSN) in Parkinson's disease and on K18 and amyloid-ß (1-42) (Aß42) in Alzheimer's disease. RR-BA showed no appreciable effect on the kinetics of K18 and Aß42 amyloid fibrillation because of their weak and non-specific interactions. However, RR-BA specifically bound to αSN with moderate binding affinity through electrostatic interactions between the positively charged RR and the negatively charged cluster in the C-terminus of αSN. In addition, hydrophobic BA in the αSN-RR-BA complex transiently condensed αSN for primary nucleation, thereby accelerating αSN amyloid fibrillation. We propose an electrostatic binding and hydrophobic condensation model of RR-BA-driven amyloid formation of αSN, which will contribute to the rational design and development of molecules for controlling amyloid aggregation in diverse fields.

Levosimendan inhibits disulfide tau oligomerization and ameliorates tau pathology in TauP301L-BiFC mice.

Tau oligomers play critical roles in tau pathology and are responsible for neuronal cell death and transmitting the disease in the brain. Accordingly, preventing tau oligomerization has become an important therapeutic strategy to treat tauopathies, including Alzheimer's disease. However, progress has been slow because detecting tau oligomers in the cellular context is difficult. Working toward tau-targeted drug discovery, our group has developed a tau-BiFC platform to monitor and quantify tau oligomerization. By using the tau-BiFC platform, we screened libraries with FDA-approved and passed phase I drugs and identified levosimendan as a potent anti-tau agent that inhibits tau oligomerization. 14C-isotope labeling of levosimendan revealed that levosimendan covalently bound to tau cysteines, directly inhibiting disulfide-linked tau oligomerization. In addition, levosimendan disassembles tau oligomers into monomers, rescuing neurons from aggregation states. In comparison, the well-known anti-tau agents methylene blue and LMTM failed to protect neurons from tau-mediated toxicity, generating high-molecular-weight tau oligomers. Levosimendan displayed robust potency against tau oligomerization and rescued cognitive declines induced by tauopathy in the TauP301L-BiFC mouse model. Our data present the potential of levosimendan as a disease-modifying drug for tauopathies.

TiO2 paste formulation for crack-free mesoporous nanocrystalline film of dye-sensitized solar cells.

Using a doctor-blade method, a highly viscous titanium dioxide (TiO2) paste was deposited on a glass substrate coated with fluorine doped tin oxide (FTO). The paste was mainly composed of commercially available TiO2 nanoparticles (P25) and hydroxypropyl cellulose (HPC) as organic filler. Varying the content of HPC in the TiO2 paste changed the physical properties of the mesoporous TiO2 layer, particularly its porosity and surface area. From the quantification of dyes on Ti2, layer and the electrochemical impedance spectroscopy (EIS) study of the dye-sensitized solar cells (DSSCs), the surface area of the TiO2 film was found to have decreased. This came with the increase of HPC content while the porosity of the film increased, consistent with the concurrent decrease of short-circuit current density (Jsc) and efficiency (eta). The increased porosity greatly affected the electron transport through the TiO2 film by decreasing the coordination number of the TiO2 particles resulting to a decrease of the electron diffusion coefficient.

Carbon nanotubes on fluorine-doped tin oxide for fabrication of dye-sensitized solar cells at low temperature condition.

The multi-walled carbon nanotubes (MWCNTs), electrophoretically deposited on fluorine-doped tin oxide (FTO), were employed as charge-collecting channels in the TiO2 photoelectrode of dye-sensitized solar cells (DSSCs) fabricated at 200 degrees C. The CNT-networks at the conducting substrate increased the charge collection efficiency of the porous TiO2 film, while the short circuit current increased up to ca. 43% under optimized condition. However, the significant decrease in the open-circuit voltage (Voc) up to ca. 132 mV resulted in the failure of the overall cell efficiency improvement. Findings reveal that the transfer process for the back electron is mainly responsible for the significant Voc drop when the MWCNTs were deposited at the electron-collecting substrate of the photoelectrode. The study demonstrates that electrophoretic deposition of MWCNTs on charge collecting substrate would be applicable to introduce an effective charge-collecting channel for the fabrication of flexible DSSCs under low temperature sintering condition.

SiReN: Sign-Aware Recommendation Using Graph Neural Networks.

In recent years, many recommender systems using network embedding (NE) such as graph neural networks (GNNs) have been extensively studied in the sense of improving recommendation accuracy. However, such attempts have focused mostly on utilizing only the information of positive user-item interactions with high ratings. Thus, there is a challenge on how to make use of low rating scores for representing users' preferences since low ratings can be still informative in designing NE-based recommender systems. In this study, we present SiReN, a new Sign-aware Recommender system based on GNN models. Specifically, SiReN has three key components: 1) constructing a signed bipartite graph for more precisely representing users' preferences, which is split into two edge-disjoint graphs with positive and negative edges each; 2) generating two embeddings for the partitioned graphs with positive and negative edges via a GNN model and a multilayer perceptron (MLP), respectively, and then using an attention model to obtain the final embeddings; and 3) establishing a sign-aware Bayesian personalized ranking (BPR) loss function in the process of optimization. Through comprehensive experiments, we empirically demonstrate that SiReN consistently outperforms state-of-the-art NE-aided recommendation methods.

The Fate of Tau Aggregates Between Clearance and Transmission.

Neuronal accumulation of mis-folded tau is the pathological hallmark of multiple neurodegenerative disorders, including Alzheimer's disease. Distinct from amyloid plaques, which appear simultaneously throughout the brain, tau pathology develops first in a specific brain region and then propagates to neuroanatomically connected brain regions, exacerbating the disease. Due to the implication in disease progression, prevention of tau transmission is recognized as an important therapeutic strategy that can halt disease progression in the brain. Recently, accumulating studies have demonstrated diverse cellular mechanisms associated with cell-to-cell transmission of tau. Once transmitted, mis-folded tau species act as a prion-like seed for native tau aggregation in the recipient neuron. In this review, we summarize the diverse cellular mechanisms associated with the secretion and uptake of tau, and highlight tau-trafficking receptors, which mediate tau clearance or cell-to-cell tau transmission.

Picomolar-sensitive ß-amyloid fibril fluorophores by tailoring the hydrophobicity of biannulated π-elongated dioxaborine-dyes.

The pathological origin of Alzheimer's disease (AD) is still shrouded in mystery, despite intensive worldwide research efforts. The selective visualization of ß-amyloid (Aß), the most abundant proteinaceous deposit in AD, is pivotal to reveal AD pathology. To date, several small-molecule fluorophores for Aß species have been developed, with increasing binding affinities. In the current work, two organic small-molecule dioxaborine-derived fluorophores were rationally designed through tailoring the hydrophobicity with the aim to enhance the binding affinity for Aß1-42 fibrils -while concurrently preventing poor aqueous solubility-via biannulate donor motifs in D-π-A dyes. An unprecedented sub-nanomolar affinity was found (K d = 0.62 ± 0.33 nM) and applied to super-sensitive and red-emissive fluorescent staining of amyloid plaques in cortical brain tissue ex vivo. These fluorophores expand the dioxaborine-curcumin-based family of Aß-sensitive fluorophores with a promising new imaging agent.