Md. Mamunul Haque

Acute toxicity and tissue distribution of CdSe/CdS-MPA quantum dots after repeated intraperitoneal injection to mice

Quantum dots (QDs) are novel tools with multiple biological and medical applications because of their superior photoemission and photostability characteristics. However, leaching of toxic metals from QDs is of great concern. Therefore, for the successful application of QDs in bioscience, it is essential to understand their biological fate and toxicity. We investigated toxicological effects and tissue distribution of mercaptopropionic acid‐conjugated cadmium selenide/cadmium sulfide (CdSe/CdS‐MPA) QDs after repeated intraperitoneal injection into BALB/c mice. The mice were injected every 3 days with various doses of QDs (0, 5, 10 and 25 mg kg−1). The subsequent effects of QDs on plasma levels of various biomarkers were evaluated at different time points (at 0, 1, 4, 7, 10, 13 and 15 days). Various tissue samples (spleen, liver, lung, kidneys, brain, heart and thymus) were collected for toxicity analysis, distribution testing, histopathological examination and inflammation assessment. No abnormal clinical signs or behaviors were recorded but the body weight of mice treated with 25 mg kg−1 QDs was significantly decreased from day 7 compared with control mice. QDs were observed in the liver, spleen, lung and kidneys, but not in brain or heart. Significantly higher levels of lactate dehydrogenase and nicotinamide adenine dinucleotide phosphate oxidase were found in the plasma, liver and spleen. Histopathological examination did not show any tissue toxicity but the levels of interleukin‐6, a pro‐inflammatory marker, were increased in the plasma, liver and spleen. All of these findings provide insight into the observed toxicological effect levels and tissue‐specific distribution of CdSe/CdS‐MPA QDs. Copyright

Bimolecular Fluorescence Complementation; Lighting-Up Tau-Tau Interaction in Living Cells

Abnormal tau aggregation is a pathological hallmark of many neurodegenerative disorders and it is becoming apparent that soluble tau aggregates play a key role in neurodegeneration and memory impairment. Despite this pathological importance, there is currently no single method that allows monitoring soluble tau species in living cells. In this regard, we developed a cell-based sensor that visualizes tau self-assembly. By introducing bimolecular fluorescence complementation (BiFC) technique to tau, we were able to achieve spatial and temporal resolution of tau-tau interactions in a range of states, from soluble dimers to large aggregates. Under basal conditions, tau-BiFC cells exhibited little fluorescence intensity, implying that the majority of tau molecules exist as monomers. Upon chemically induced tau hyperphosphorylation, BiFC fluorescence greatly increased, indicating an increased level of tau-tau interactions. As an indicator of tau assembly, our BiFC sensor would be a useful tool for investigating tau pathology.

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.

Photo-affinity labeling (PAL) in chemical proteomics: a handy tool to investigate protein-protein interactions (PPIs)

Protein-protein interactions (PPIs) trigger a wide range of biological signaling pathways that are crucial for biomedical research and drug discovery. Various techniques have been used to study specific proteins, including affinity chromatography, activity-based probes, affinity-based probes and photo-affinity labeling (PAL). PAL has become one of the most powerful strategies to study PPIs. Traditional photocrosslinkers are used in PAL, including benzophenone, aryl azide, and diazirine. Upon photoirradiation, these photocrosslinkers (Pls) generate highly reactive species that react with adjacent molecules, resulting in a direct covalent modification. This review introduces recent examples of chemical proteomics study using PAL for PPIs.

In vitro screening of NADPH oxidase inhibitors and in vivo effects of L-leucinethiol on experimental autoimmune encephalomyelitis-induced mice

Experimental autoimmune encephalomyelitis (EAE), a Th1 polarized demyelinating disease of the central nervous system, shares many pathological and clinical similarities with multiple sclerosis (MS). The objectives of this study were i) to evaluate the suppressive effects of L-leucinethiol (LeuSH), a metalloprotease inhibitor on EAE-induced mice and ii) to study the effects of LeuSH on matrix metalloproteinase-9 (MMP-9), NADPH oxidase and cytokines (IFN-γ, IL-5 and IL-10) in tissues and plasma of EAE mice as a measure of potential markers associated with EAE disease. C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein (MOG35-55) peptide in complete Freunds adjuvant to induce EAE. A significant difference was observed in body weights and clinical signs of LeuSH (8 mg/kg) administered EAE-induced mice compared to control mice. The findings of this study include alterations in the enzymatic expression of MMP-9, NADPH oxidase and cytokine levels in the brain, spinal cord, spleen, thymus and plasma of inhibitor-treated EAE mice as well as EAE-induced mice. The enzyme activities of NADPH oxidase were inhibited by LeuSH. From these results, it can be considered that LeuSH acts as one of the antigen candidates in ameliorating the clinical symptoms of EAE disease in mice.

Chloro‐functionalized photo‐crosslinking BODIPY for glutathione sensing and sub‐cellular trafficking

Glutathione (GSH) is one of major antioxidants inside cells that regulates oxidoreduction homeostasis. Recently, there have been extensive efforts to visualize GSH in live cells, but most of the probes available today are simple detection sensors and do not provide details of cellular localization. A new fluorescent probe (pcBD2‐Cl), which is cell permeable and selectively reacts with GSH in situ, has been developed. The in situ GSH‐labeled probe (pcBD2–GSH) exhibited quenches fluorescence, but subsequent binding to cellular abundant glutathione S‐transferase (GST) recovers the fluorescence intensity, which makes it possible to image the GSH–GST complex in live cells. Interactions between probe and GST were confirmed by means of photo‐crosslinking under intact live‐cell conditions. Interestingly, isomers of chloro‐functionalized 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) compounds behaved very distinctively inside the cells. Following co‐staining imaging with MitoTracker and mitochondria fractionation upon lipopolysaccharide‐mediated reactive oxygen species induction experiments showed that pcBD2–GSH accumulated in mitochondria. This is the first example of a live‐cell imaging probe to visualize translocation of GSH from the cytosol to mitochondria.

Discrimination of Avian Influenza Virus Subtypes using Host‐Cell Infection Fingerprinting by a Sulfinate‐based Fluorescence Superoxide Probe

The current gold-standard diagnosis method for avian influenza (AI) is an embryonic egg-based hemagglutination assay followed by immunoblotting or PCR sequencing to confirm subtypes. It requires, however, specialized facilities to handle egg inoculation and incubation, and the subtyping methods relied on costly reagents. Now, the first differential sensing approach to distinguish AI subtypes is demonstrated using series of cell lines and a fluorescent sensor. Susceptibility of AI virus differs depending on genetic backgrounds of host cells. Cells were examined from different organ origins, and the infection patterns against a panel of cells were utilized for AI virus subtyping. To quantify AI infection, a highly cell-permeable fluorescent superoxide sensor was designed to visualize infection. This differential sensing strategy successfully proved discriminations of AI subtypes and demonstrated as a useful primary screening platform to monitor a large number of samples.