Sungsu Lim

Identification of disulfide cross-linked tau dimer responsible for tau propagation

Recent evidence suggests that tau aggregates are not only neurotoxic, but also propagate in neurons acting as a seed for native tau aggregation. Prion-like tau transmission is now considered as an important pathogenic mechanism driving the progression of tau pathology in the brain. However, prion-like tau species have not been clearly characterized. To identify infectious tau conformers, here we prepared diverse tau aggregates and evaluated the effect on inducing intracellular tau-aggregation. Among tested, tau dimer containing P301L-mutation is identified as the most infectious form to induce tau pathology. Biochemical analysis reveals that P301L-tau dimer is covalently cross-linked with a disulfide bond. The relatively small and covalently cross-linked tau dimer induced tau pathology efficiently in primary neurons and also in tau-transgenic mice. So far, the importance of tau disulfide cross-linking has been overlooked in the study of tau pathology. Here our results suggested that tau disulfide cross-linking might play critical role in tau propagation by producing structurally stable and small tau conformers.

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 β-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.

Inhibition of tau aggregation by a rosamine derivative that blocks tau intermolecular disulfide cross-linking

Abstract Abnormal tau aggregates are presumed to be neurotoxic and are an important therapeutic target for multiple neurodegenerative disorders including Alzheimer’s disease. Growing evidence has shown that tau intermolecular disulfide cross-linking is critical in generating tau oligomers that serve as a building block for higher-order aggregates. Here we report that a small molecule inhibitor prevents tau aggregation by blocking the generation of disulfide cross-linked tau oligomers. Among the compounds tested, a rosamine derivative bearing mild thiol reactivity selectively labeled tau and effectively inhibited oligomerization and fibrillization processes in vitro. Our data suggest that controlling tau oxidation status could be a new therapeutic strategy for prevention of abnormal tau aggregation.

Cell-based Models To Investigate Tau Aggregation

Accumulation of abnormal tau aggregates in neuron is an important pathological signature in multiple neurodegenerative disorders including Alzheimers disease. Tau is a neuron specific microtubule-associated protein that regulates microtubule stability, which is critical for axonal outgrowth and synaptic plasticity. In a pathological condition, tau dissociates from microtubules and forms insoluble aggregates called neurofibrillary tangles (NFTs). The accumulation of NFTs in neuron directly correlates with microtubule dysfunction and neuronal degeneration. Due to the pathophysiological importance of tau, great efforts have been made to understand tau aggregation processes and find therapeutics to halt or reverse the processes. However, progress has been slow due to the lack of a suitable method for monitoring tau aggregation. In this mini-review, we will review the conventional methods for studying tau aggregation, and introduce recent cell-based sensor approaches that allow monitoring tau aggregation in living cells.

Transcriptome analyses of chronic traumatic encephalopathy show alterations in protein phosphatase expression associated with tauopathy

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disorder that is associated with repetitive head injury and has distinctive neuropathological features that differentiate this disease from other neurodegenerative diseases. Intraneuronal tau aggregates, although they occur in different patterns, are diagnostic neuropathological features of CTE, but the precise mechanism of tauopathy is not known in CTE. We performed whole RNA sequencing analysis of post-mortem brain tissue from patients with CTE and compared the results to normal controls to determine the transcriptome signature changes associated with CTE. The results showed that the genes related to the MAP kinase and calcium-signaling pathways were significantly downregulated in CTE. The altered expression of protein phosphatases (PPs) in these networks further suggested that the tauopathy observed in CTE involves common pathological mechanisms similar to Alzheimer’s disease (AD). Using cell lines and animal models, we also showed that reduced PPP3CA/PP2B phosphatase activity is directly associated with increases in phosphorylated (p)-tau proteins. These findings provide important insights into PP-dependent neurodegeneration and may lead to novel therapeutic approaches to reduce the tauopathy associated with CTE.

Glioblastoma-secreted soluble CD44 activates tau pathology in the brain

During aggressive tumor growth and migration, glioblastoma cells secrete diverse molecules and adhesion proteins to the extracellular matrix. Yet, the biochemical effects of the glioblastoma secretome in the brain remain largely unknown. Here we show that soluble CD44 secreted from glioblastoma cells induces neuronal degeneration through the activation of tau pathology in the brain. Glioblastoma-xenograft tissues showed a number of degenerating neurons bearing highly phosphorylated tau. Through a series of secretome-analyses, we identified that soluble CD44 was the responsible protein inducing tau phosphorylation and aggregation (EC50u2009=u200919.1u2009ng/mL). The treatment of sCD44 to primary hippocampal neurons-induced tau hyperphosphorylation, leading to neuronal degeneration. Also, the injection of sCD44 into the brains of tau transgenic mice induced tau hyper-phosphorylation in hippocampal neurons. Altogether, our data suggest a neurodegenerative role of sCD44 in promoting tau pathology and serving as a molecular link between glioblastoma and neurodegeneration.Brain tumors: Protein prompt for brain degenerationA protein secreted by aggressive brain tumors triggers the degeneration of neurons in surrounding brain tissues. The most aggressive brain tumors are formed by glioblastoma cells, which secrete molecules that infiltrate surrounding brain tissues, leading to loss of memory, communication and motor functions. Researchers led by Cheolju Lee and Yun Kyung Kim at the Korea Institute of Science and Technology in Seoul have shown that the CD44 protein, secreted by glioblastoma cells, is responsible for triggering this neurodegeneration. They discovered that sCD44 activates another process known as tau pathology, which is characteristic of multiple neuro-degnerative disorders such as Alzheimer’s disease. The tau protein usually stabilizes internal cellular structures, but when it is modified by abnormal activity such as the elevated levels of sCD44 found in this study, it forms insoluble masses, disrupting neuronal structure and function.

Nootropic nanocomplex with enhanced blood-brain barrier permeability for treatment of traumatic brain injury-associated neurodegeneration

ABSTRACT Traumatic brain injury (TBI) is an intracranial injury which can induce immediate neuroinflammation and long‐term neurological deficits. Methylene blue (MB) as a nootropic has a great potential to treat neurodegeneration after TBI because of its anti‐inflmmatory and neuroprotective functions. However, its limited accumulation to the brain across the blood‐brain barrier (BBB) remains a major hurdle to be overcome. In this paper, we present a polymer surfactant‐encapsulated nanocomplex of MB as a delivery system with high BBB permeability for efficacious treatment of TBI‐induced neurodegeneration. MB was formulated via electrostatically/hydrophobically directed assembly with fatty acid and Pluronic surfactant (F‐127 or F‐68) to construct nanocomplexes of two different colloidal sizes (<10 nm and ˜108 nm in hydrodynamic diameter for NanoMB‐127 and NanoMB‐68, respectively). Compared to uncomplexed free MB, formulation into the ultrasmall nanocomplex (NanoMB‐127) significantly enhanced the uptake of MB by blood‐brain vascular endothelial bEnd3 cells in vitro, and indeed improved its BBB penetration upon systemic administration to normal mice in vivo. However, large‐size NanoMB‐68 showed negligible BBB crossing despite the efficient bEnd3 cell internalization in vitro, probably due to the unfavorable pharmacokinetic profile associated with its large particle size. By virtue of the efficient BBB penetration and cellular uptake, ultrasmall NanoMB‐127 was shown to distinctively reduce the expression level of an inflammatory cytokine with no notable toxicity in vitro and also considerably prevent the neurodegeneration after TBI in mice at much lower doses than free MB. Overall, the Pluronic‐supported nanocomplexation method allows efficient brain delivery of MB, offering a novel way of enhancing the efficacy of neurotherapeutics to treat brain diseases. Graphical abstract Figure. No caption available.

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.

A guanidine-appended scyllo -inositol derivative AAD-66 enhances brain delivery and ameliorates Alzheimer’s phenotypes

Alzheimer’s disease (AD) is a degenerative brain disease that destroys memory and other important mental functions but lacks efficient therapeutic agents. Blocking toxic amyloid β (Aβ) could be beneficial for AD and represents a promising therapeutic strategy for AD treatment. scyllo-Inositol (SI) is a potential therapeutic for AD by directly interacting with the Aβ peptide to inhibit Aβ42 fiber formation. Clinical studies of SI showed promising benefits on mild to moderate AD, however, with limitations on dosage regime. A new strategy to enhance the brain delivery of SI is needed to achieve the efficacy with minimum adverse effects. Herein, we report that a novel guanidine-appended SI derivative AAD-66 resulted in more effective reductions of brain Aβ and plaque deposits, gliosis, and behavioral memory deficits in the disease-established 5xFAD mice. Overall, our present study reveals the potential of AAD-66 as a promising therapeutic agent for AD.

Image-Based Analysis of Intracellular Tau Aggregation by Using Tau-BiFC Cell Model.

Abnormal tau aggregation is a pathological hallmark of neurodegenerative disease classified as tauopathy. Preventing tau aggregation becomes an important therapeutic strategy to cure tau-mediated neurodegeneration. Here, we describe a method to investigate intracellular tau aggregation by using a recently developed tau aggregation cell-based model named tau-BiFC. High-throughput and high-contents screening method for quantifying intracellular tau aggregation would expedite the discovery of drugs that inhibit tau aggregation.