Chaeyoung Kim

HDAC6 Inhibitor Blocks Amyloid Beta-Induced Impairment of Mitochondrial Transport in Hippocampal Neurons

Even though the disruption of axonal transport is an important pathophysiological factor in neurodegenerative diseases including Alzheimers disease (AD), the relationship between disruption of axonal transport and pathogenesis of AD is poorly understood. Considering that α-tubulin acetylation is an important factor in axonal transport and that Aβ impairs mitochondrial axonal transport, we manipulated the level of α-tubulin acetylation in hippocampal neurons with Aβ cultured in a microfluidic system and examined its effect on mitochondrial axonal transport. We found that inhibiting histone deacetylase 6 (HDAC6), which deacetylates α-tubulin, significantly restored the velocity and motility of the mitochondria in both anterograde and retrograde axonal transports, which would be otherwise compromised by Aβ. The inhibition of HDAC6 also recovered the length of the mitochondria that had been shortened by Aβ to a normal level. These results suggest that the inhibition of HDAC6 significantly rescues hippocampal neurons from Aβ-induced impairment of mitochondrial axonal transport as well as mitochondrial length. The results presented in this paper identify HDAC6 as an important regulator of mitochondrial transport as well as elongation and, thus, a potential target whose pharmacological inhibition contributes to improving mitochondrial dynamics in Aβ treated neurons.

Cancer gene therapy using a novel secretable trimeric TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, is a type II transmembrane cytokine molecule. Soluble TRAIL has been shown to induce apoptosis in a wide variety of cancer cells in vitro and to suppress tumor growth specifically without damaging normal cells and tissues in vivo. In our previous report, we have demonstrated that an artificial gene encoding the polypeptide composed of the three functional elements (a secretion signal, a trimerization domain and an apoptosis-inducing moiety of TRAIL gene sequence) expresses and secretes highly apoptotic trimeric TRAIL into the culture supernatant. Here, as an approach to TRAIL-based cancer gene therapy, we developed an adenoviral vector delivering the gene that encodes our secretable trimeric TRAIL (stTRAIL). This adenovirus (Ad-stTRAIL) potently induced apoptosis in vitro in cancer cell lines such as HeLa, MDA-MB-231, A549, HCT116 and U-87MG. In an animal xenograft tumor model bearing a human glioma cell line U-87MG, intratumoral delivery of Ad-stTRAIL dramatically suppressed tumor growth without showing detectable adverse side effects. Histological analysis revealed that Ad-stTRAIL suppresses tumor growth by inducing apoptotic cell death. Contrary to the known rapid clearance of systemically delivered TRAIL protein from the blood circulation, stTRAIL expressed by Ad-stTRAIL in tumor tissues persisted for more than 4 days. In a comparison of tumor suppressor activity between Ad-stTRAIL and Ad-flTRAIL (delivering the full-length TRAIL gene) after mixing infected cells with uninfected cells and implanting these mixed cells in nude mice, Ad-stTRAIL showed higher tumor suppressor activity than that of Ad-flTRAIL. Our data reveal that a gene therapy using Ad-stTRAIL has a promising potential to treat human cancers including gliomas.

Possible Novel Therapy for Malignant Gliomas with Secretable Trimeric TRAIL

Malignant gliomas are the most common primary brain tumors. Despite intensive clinical investigation and many novel therapeutic approaches, average survival for the patients with malignant gliomas is only about 1 year. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown potent and cancer-selective killing activity and drawn considerable attention as a promising therapy for cancers, but concerns over delivery and toxicity have limited progress. We have developed a secretable trimeric TRAIL (stTRAIL) and here evaluated the therapeutic potential of this stTRAIL-based gene therapy in brain tumors. An adenovirus (Ad-stTRAIL) delivering stTRAIL was injected into intra-cranial human glioma tumors established in nude mice and tumor growth monitored using the magnetic resonance imaging (MRI). Ad-stTRAIL gene therapy showed potent tumor suppressor activity with no toxic side effects at therapeutically effective doses. When compared with 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a conventional therapy for malignant gliomas, Ad-stTRAIL suppressed tumor growth more potently. The combination of Ad-stTRAIL and BCNU significantly increased survival compared to the control mice or mice receiving Ad-stTRAIL alone. Our data indicate that Ad-stTRAIL, either alone or combined with BCNU, has promise as a novel therapy for malignant gliomas.

Acute ER stress regulates amyloid precursor protein processing through ubiquitin-dependent degradation

Beta-amyloid (Aβ), a major pathological hallmark of Alzheimers disease (AD), is derived from amyloid precursor protein (APP) through sequential cleavage by β-secretase and γ-secretase enzymes. APP is an integral membrane protein, and plays a key role in the pathogenesis of AD; however, the biological function of APP is still unclear. The present study shows that APP is rapidly degraded by the ubiquitin-proteasome system (UPS) in the CHO cell line in response to endoplasmic reticulum (ER) stress, such as calcium ionophore, A23187, induced calcium influx. Increased levels of intracellular calcium by A23187 induces polyubiquitination of APP, causing its degradation. A23187-induced reduction of APP is prevented by the proteasome inhibitor MG132. Furthermore, an increase in levels of the endoplasmic reticulum-associated degradation (ERAD) marker, E3 ubiquitin ligase HRD1, proteasome activity, and decreased levels of the deubiquitinating enzyme USP25 were observed during ER stress. In addition, we found that APP interacts with USP25. These findings suggest that acute ER stress induces degradation of full-length APP via the ubiquitin-proteasome proteolytic pathway.

Analysis of Extracellular RNA by Digital PCR

The transfer of extracellular RNA is emerging as an important mechanism for inter-cellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

miR-27a and miR-27b regulate autophagic clearance of damaged mitochondria by targeting PTEN-induced putative kinase 1 (PINK1)

BackgroundLoss-of-function mutations in PINK1 and PARKIN are the most common causes of autosomal recessive Parkinson’s disease (PD). PINK1 is a mitochondrial serine/threonine kinase that plays a critical role in mitophagy, a selective autophagic clearance of damaged mitochondria. Accumulating evidence suggests mitochondrial dysfunction is one of central mechanisms underlying PD pathogenesis. Therefore, identifying regulatory mechanisms of PINK1 expression may provide novel therapeutic opportunities for PD. Although post-translational stabilization of PINK1 upon mitochondrial damage has been extensively studied, little is known about the regulation mechanism of PINK1 at the transcriptional or translational levels.ResultsHere, we demonstrated that microRNA-27a (miR-27a) and miR-27b suppress PINK1 expression at the translational level through directly binding to the 3′-untranslated region (3′UTR) of its mRNA. Importantly, our data demonstrated that translation of PINK1 is critical for its accumulation upon mitochondrial damage. The accumulation of PINK1 upon mitochondrial damage was strongly regulated by expression levels of miR-27a and miR-27b. miR-27a and miR-27b prevent mitophagic influx by suppressing PINK1 expression, as evidenced by the decrease of ubiquitin phosphorylation, Parkin translocation, and LC3-II accumulation in damaged mitochondria. Consequently, miR-27a and miR-27b inhibit lysosomal degradation of the damaged mitochondria, as shown by the decrease of the delivery of damaged mitochondria to lysosome and the degradation of cytochrome c oxidase 2 (COX2), a mitochondrial marker. Furthermore, our data demonstrated that the expression of miR-27a and miR-27b is significantly induced under chronic mitophagic flux, suggesting a negative feedback regulation between PINK1-mediated mitophagy and miR-27a and miR-27b.ConclusionsWe demonstrated that miR-27a and miR-27b regulate PINK1 expression and autophagic clearance of damaged mitochondria. Our data further support a novel negative regulatory mechanism of PINK1-mediated mitophagy by miR-27a and miR-27b. Therefore, our results considerably advance our understanding of PINK1 expression and mitophagy regulation and suggest that miR-27a and miR-27b may represent potential therapeutic targets for PD.

Synapsin-1 and tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes.

O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, β-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, 603QASQAGPGPR612, and the serine residue at 692 of the tau peptide, 688SPVVSGDTSPR698, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.

Pharmacokinetics and biodistribution of a pGT2-VEGF plasmid DNA after administration in rats.

Intramyocardial administration of gene therapy vectors expressing angiogenic factors have been attempted as an alternative to conventional surgical methods for the management of myocardial ischemia. In this study, we have developed the pGT2-VEGF, a plasmid DNA vector expressing human VEGF165, for the management of ischemic cardiovascular disease and investigated in vivo pharmacokinetics and tissue distribution of pGT2-VEGF after intramyocardial and intravenous administration in rats. A high concentration of pGT2-VEGF was observed in the heart after intramyocardial injection of 300 μg, which is in line with the assumption that direct intramyocardial delivery enables extended localization at the administration site. Leakage of the pGT2-VEGF to the blood circulation was observed after intramyocardial injection, with an area under the curve (AUC) of 3.8 μg min/mL, as compared with 37.3 μg min/mL after intravenous injection of the same dose. The pGT2-VEGF concentration in blood peaked at 5 minutes after intramyocardial administration and declined rapidly to undetectable levels by 2 hours post-administration. In tissue distribution studies, pGT2-VEGF peaked at 5 minutes post-administration in various organs but was undetectable at 2 hours in all organs except heart, lung, and liver. Taken together, the results suggest that intramyocardial-delivered pGT2-VEGF was degraded rapidly in vivo and mainly persisted in target tissues, the heart. In addition, intramyocardial-administered pGT2-VEGF was expressed for longer periods than the persistence of the pGT2-VEGF plasmid DNA in a target tissue. Therefore, a direct myocardial injection of pGT2-VEGF might be useful for local therapeutic angiogenesis.

Preclinical studies for pharmacokinetics and biodistribution of Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy

Malignant glioma is the most frequent type in brain tumors. The prognosis of this tumor has not been significantly improved for the past decades and the average survival of patients is less than one year. Thus, an effective novel therapy is urgently needed. TNF-related apoptosis inducing ligand (TRAIL), known to have tumor cell-specific killing activity, has been investigated as a novel therapeutic for cancers. We have developed Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy and demonstrated the potential to treat malignant gliomas. Currently, this Ad-stTRAIL gene therapy is under phase I clinical trial for malignant gliomas. Here, we report preclinical studies for Ad-stTRAIL carried out using rats. We delivered Ad-stTRAIL intracranially and determined its pharmacokinetics and biodistribution. Most Ad-stTRAIL remained in the delivered site and the relatively low number of viral genomes was detected in the opposite site of brain and cerebrospinal fluid. Similarly, only small portion of the viral particles injected was found in the blood plasma and major organs and tissues, probably due to the brain-blood barrier. Multiple administrations did not lead to accumulation of Ad-stTRAIL at the injection site and organs. Repeated delivery of Ad-stTRAIL did not show any serious side effects. Our data indicate that intracranially delivered Ad-stTRAIL is a safe approach, demonstrating the potential as a novel therapy for treating gliomas.

Common Pesticide, Dichlorodiphenyltrichloroethane (DDT), Increases Amyloid-β Levels by Impairing the Function of ABCA1 and IDE: Implication for Alzheimer's Disease.

While early-onset familial Alzheimers disease (AD) is caused by a genetic mutation, the vast majority of late-onset AD is likely caused by the combination of genetic and environmental factors. Unlike genetic studies, potential environmental factors affecting AD pathogenesis have not yet been thoroughly investigated. Among environmental factors, pesticides seem to be one of critical environmental contributors to late-onset AD. Recent studies reported that the serum and brains of AD patients have dramatically higher levels of a metabolite of dichlorodiphenyltrichloroethane (DDT). While these epidemiological studies provided initial clues to the environmental risks potentially contributing to disease pathogenesis, a functional approach is required to determine whether they actually have a causal role in disease development. In our study, we addressed this critical knowledge gap by investigating possible mechanisms by which DDT affects amyloid-β (Aβ) levels. We treated H4-AβPPswe or H4 cells with DDT to analyze its effect on Aβ metabolism using Aβ production, clearance, and degradation assays. We found that DDT significantly increased the levels of amyloid-β protein precursor (AβPP) and β-site AβPP-cleaving enzyme1 (BACE1), affecting Aβ synthesis pathway in H4-AβPPswe cells. Additionally, DDT impaired the clearance and extracellular degradation of Aβ peptides. Most importantly, we identified for the first time that ATP-binding cassette transporter A1 (ABCA1) and insulin-degrading enzyme (IDE) are the downstream target genes adversely affected by DDT. Our findings provide insight into the molecular mechanisms by which DDT exposure may increase the risk of AD, and it further supports that ABCA1 and IDE may be potential therapeutic targets.