Bonghee Lee

The first Korean genome sequence and analysis: Full genome sequencing for a socio-ethnic group

We present the first Korean individual genome sequence (SJK) and analysis results. The diploid genome of a Korean male was sequenced to 28.95-fold redundancy using the Illumina paired-end sequencing method. SJK covered 99.9% of the NCBI human reference genome. We identified 420,083 novel single nucleotide polymorphisms (SNPs) that are not in the dbSNP database. Despite a close similarity, significant differences were observed between the Chinese genome (YH), the only other Asian genome available, and SJK: (1) 39.87% (1,371,239 out of 3,439,107) SNPs were SJK-specific (49.51% against Venters, 46.94% against Watsons, and 44.17% against the Yoruba genomes); (2) 99.5% (22,495 out of 22,605) of short indels (< 4 bp) discovered on the same loci had the same size and type as YH; and (3) 11.3% (331 out of 2920) deletion structural variants were SJK-specific. Even after attempting to map unmapped reads of SJK to unanchored NCBI scaffolds, HGSV, and available personal genomes, there were still 5.77% SJK reads that could not be mapped. All these findings indicate that the overall genetic differences among individuals from closely related ethnic groups may be significant. Hence, constructing reference genomes for minor socio-ethnic groups will be useful for massive individual genome sequencing.

Amyloid β-protein (Aβ)1–40 protects neurons from damage induced by Aβ1–42 in culture and in rat brain

Previously, we found that amyloid β‐protein (Aβ)1–42 exhibits neurotoxicity, while Aβ1–40 serves as an antioxidant molecule by quenching metal ions and inhibiting metal‐mediated oxygen radical generation. Here, we show another neuroprotective action of nonamyloidogenic Aβ1–40 against Aβ1–42‐induced neurotoxicity in culture and in vivo. Neuronal death was induced by Aβ1–42 at concentrations higher than 2 μm, which was prevented by concurrent treatment with Aβ1–40 in a dose‐dependent manner. However, metal chelators did not prevent Aβ1–42‐induced neuronal death. Circular dichroism spectroscopy showed that Aβ1–40 inhibited the β‐sheet transformation of Aβ1–42. Thioflavin‐T assay and electron microscopy analysis revealed that Aβ1–40 inhibited the fibril formation of Aβ1–42. In contrast, Aβ1–16, Aβ25–35, and Aβ40–1 did not inhibit the fibril formation of Aβ1–42 nor prevent Aβ1–42‐induced neuronal death. Aβ1–42 injection into the rat entorhinal cortex (EC) caused the hyperphosphorylation of tau on both sides of EC and hippocampus and increased the number of glial fibrillary acidic protein (GFAP)‐positive astrocytes in the ipsilateral EC, which were prevented by the concurrent injection of Aβ1–40. These results indicate that Aβ1–40 protects neurons from Aβ1–42‐induced neuronal damage in vitro and in vivo, not by sequestrating metals, but by inhibiting the β‐sheet transformation and fibril formation of Aβ1–42. Our data suggest a mechanism by which elevated Aβ1–42/Aβ1–40 ratio accelerates the development of Alzheimers disease (AD) in familial AD.

A Systems Approach for Decoding Mitochondrial Retrograde Signaling Pathways

Transcriptional profiling and transcription factor matching reveal potential targets for therapies against diseases associated with mitochondrial dysfunction. Signaling Mitochondrial Dysfunction The mitochondrial and nuclear genomes contribute to mitochondrial function, and when mitochondrial function is compromised, mitochondrial retrograde signaling alters nuclear gene expression. Chae et al. performed gene expression profiling of engineered cells that had mitochondria containing a disease-associated mutation that causes mitochondrial dysfunction. By generating networks of transcription factors that targeted these genes, the authors revealed putative mitochondrial retrograde signaling pathways. One such pathway involved retinoic X receptor α (RXRA), the mRNA for which was reduced in the mutant cells. Network analysis and experiments in cells suggested that mitochondrial dysfunction caused by the mutation initiated a positive feedback loop that aggravated mitochondrial dysfunction: Reduced RXRA abundance further compromised expression of genes encoding products involved in mitochondrial function and translation. This gene transcription factor mapping network approach may reveal targets for therapeutic intervention of diseases associated with mitochondrial dysfunction. Mitochondrial dysfunctions activate retrograde signaling from mitochondria to the nucleus. To identify transcription factors and their associated pathways that underlie mitochondrial retrograde signaling, we performed gene expression profiling of the cells engineered to have varying amounts of mitochondrial DNA with an A3243G mutation (mt3243) in the leucine transfer RNA (tRNALeu), which reduces the abundance of proteins involved in oxidative phosphorylation that are encoded by the mitochondrial genome. The cells with the mutation exhibited reduced mitochondrial function, including compromised oxidative phosphorylation, which would activate diverse mitochondrial retrograde signaling pathways. By analyzing the gene expression profiles in cells with the mutant tRNALeu and the transcription factors that recognize the differentially regulated genes, we identified 72 transcription factors that were potentially involved in mitochondrial retrograde signaling. We experimentally validated that the mt3243 mutation induced a retrograde signaling pathway involving RXRA (retinoid X receptor α), reactive oxygen species, kinase JNK (c-JUN N-terminal kinase), and transcriptional coactivator PGC1α (peroxisome proliferator–activated receptor γ, coactivator 1 α). This RXR pathway contributed to the decrease in mRNA abundances of oxidative phosphorylation enzymes encoded in the nuclear genome, thereby aggravating the dysfunction in oxidative phosphorylation caused by the reduced abundance of mitochondria-encoded enzymes of oxidative phosphorylation. Thus, matching transcription factors to differentially regulated gene expression profiles was an effective approach to understand mitochondrial retrograde signaling pathways and their roles in mitochondrial dysfunction.

Proteomic Analysis of Exosomes from Human Neural Stem Cells by Flow Field-Flow Fractionation and Nanoflow Liquid Chromatography-Tandem Mass Spectrometry

Exosomes, small membrane vesicles secreted by a multitude of cell types, are involved in a wide range of physiological roles such as intercellular communication, membrane exchange between cells, and degradation as an alternative to lysosomes. Because of the small size of exosomes (30-100 nm) and the limitations of common separation procedures including ultracentrifugation and flow cytometry, size-based fractionation of exosomes has been challenging. In this study, we used flow field-flow fractionation (FlFFF) to fractionate exosomes according to differences in hydrodynamic diameter. The exosome fractions collected from FlFFF runs were examined by transmission electron microscopy (TEM) to morphologically confirm their identification as exosomes. Exosomal lysates of each fraction were digested and analyzed using nanoflow LC-ESI-MS-MS for protein identification. FIFFF, coupled with mass spectrometry, allows nanoscale size-based fractionation of exosomes and is more applicable to primary cells and stem cells since it requires much less starting material than conventional gel-based separation, in-gel digestion and the MS-MS method.

The dopamine D2 receptor regulates the development of dopaminergic neurons via extracellular signal-regulated kinase and Nurr1 activation.

Because the dopaminergic pathways in the midbrain have been closely associated with serious neuropsychiatric disorders, the elucidation of the mechanisms underlying dopaminergic neuronal development should provide some important clues for related disorders. In mice lacking the dopamine D2 receptor (D2R−/−), stereological cell counting analysis showed that the number of mesencephalic tyrosine hydroxylase (TH) cells was significantly low during ontogeny, compared with that observed in wild-type (WT) mice, thereby indicating an alteration in dopaminergic neuronal development in the absence of D2R. The results of immunohistochemical and reverse transcription-PCR analyses revealed that the expression of Nurr1, an orphan nuclear receptor, as well as Ptx3 expression, was selectively reduced in D2R−/− mice during the embryonic stage. A reporter gene assay using the Nur response element linked to the luciferase reporter gene indicated that the stimulation of D2R results in the activation of the Nurr1-mediated reporter gene. This D2R-mediated Nur response element-dependent transcriptional activity was regulated via the activation of extracellular signal-regulated kinase (ERK). Furthermore, quinpirole treatment was shown to elicit an increase in the number of TH-positive neurons, as well as the neuritic extension of TH neurons, coupled with ERK activation and Nurr1 activation in the TH-positive neurons in primary mesencephalic cultures from WT mice. However, this regulation was not detected in the D2R−/− mice. These results suggest that signaling through D2R in association with Nurr1 using ERK, plays a critical role in mesencephalic dopaminergic neuronal development.

Protein networks markedly improve prediction of subcellular localization in multiple eukaryotic species

The function of a protein is intimately tied to its subcellular localization. Although localizations have been measured for many yeast proteins through systematic GFP fusions, similar studies in other branches of life are still forthcoming. In the interim, various machine-learning methods have been proposed to predict localization using physical characteristics of a protein, such as amino acid content, hydrophobicity, side-chain mass and domain composition. However, there has been comparatively little work on predicting localization using protein networks. Here, we predict protein localizations by integrating an extensive set of protein physical characteristics over a proteins extended protein–protein interaction neighborhood, using a classification framework called ‘Divide and Conquer k-Nearest Neighbors’ (DC-kNN). These predictions achieve significantly higher accuracy than two well-known methods for predicting protein localization in yeast. Using new GFP imaging experiments, we show that the network-based approach can extend and revise previous annotations made from high-throughput studies. Finally, we show that our approach remains highly predictive in higher eukaryotes such as fly and human, in which most localizations are unknown and the protein network coverage is less substantial.

Effects of fluoxetine on ischemic cells and expressions in BDNF and some antioxidants in the gerbil hippocampal CA1 region induced by transient ischemia

Fluoxetine, a selective serotonin reuptake inhibitor, alters several physiological processes, for example, elevating intracellular cAMP level, in the hippocampus. We examined the effect of fluoxetine on ischemia-induced neuronal death, the expression of brain-derived neurotrophic factor (BDNF) and changes in some antioxidative enzymes in the hippocampal CA1 region induced by transient ischemia. In addition, we also studied the effect of fluoxetine on locomotor activity in gerbils after ischemia/reperfusion. Animals were administered with various doses of fluoxetine (10, 20, and 40 mg/kg, i.p.) once daily for 3 days before the ischemic surgery. The treatment of 10 mg/kg and 20 mg/kg fluoxetine did not show significant neuroprotective effects on CA1 pyramidal cells 4 days after ischemia/reperfusion, while the treatment with 40 mg/kg fluoxetine in ischemic animals showed about 77% neuronal survival rate compared to the control group. The treatment of 40 mg/kg fluoxetine in ischemic animals enhanced significantly BDNF, catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase-1 (SOD1) immunoreactivity in the CA1 region compared to those in the saline-treated group 4 days after ischemia/reperfusion. In addition, the treatment of fluoxetine (10, 20, 40 mg/kg) significantly inhibited post-ischemic hyperactivity. In brief, treatment with fluoxetine protects neuronal damage after transient ischemia, and the neuroprotective effect of fluoxetine in an ischemic animal model may be related with the up-regulation of BDNF, CAT, GPX, and SOD1 expression.

Human Microglial Cells Synthesize Albumin in Brain

Albumin, an abundant plasma protein with multifunctional properties, is mainly synthesized in the liver. Albumin has been implicated in Alzheimers disease (AD) since it can bind to and transport amyloid beta (Aβ), the causative agent of AD; albumin is also a potent inhibitor of Aβ polymerization. Despite evidence of non-hepatic transcription of albumin in many tissues including kidney and pancreas, non-hepatic synthesis of albumin at the protein level has been rarely confirmed. In a pilot phase study of Human Brain Proteome Project, we found evidence that microglial cells in brain may synthesize albumin. Here we report, for the first time, the de novo synthesis of albumin in human microglial cells in brain. Furtherore, we demonstrate that the synthesis and secretion of albumin from microglial cells is enhanced upon microgial activation by Aβ1–42- or lipopolysaccharide (LPS)-treatment. These data indicate that microglial cells may play a beneficial role in AD by secreting albumin that not only inhibits Aβ polymerization but also increases its clearance.

Rho-kinase regulates energy balance by targeting hypothalamic leptin receptor signaling

Leptin regulates energy balance. However, knowledge of the critical intracellular transducers of leptin signaling remains incomplete. We found that Rho-kinase 1 (ROCK1) regulates leptin action on body weight homeostasis by activating JAK2, an initial trigger of leptin receptor signaling. Leptin promoted the physical interaction of JAK2 and ROCK1, thereby increasing phosphorylation of JAK2 and downstream activation of Stat3 and FOXO1. Mice lacking ROCK1 in either pro-opiomelanocortin (POMC) or agouti-related protein neurons, mediators of leptin action, displayed obesity and impaired leptin sensitivity. In addition, deletion of ROCK1 in the arcuate nucleus markedly enhanced food intake, resulting in severe obesity. Notably, ROCK1 was a specific mediator of leptin, but not insulin, regulation of POMC neuronal activity. Our data identify ROCK1 as a key regulator of leptin action on energy homeostasis.

Mitochondrial dysfunction and tissue injury by alcohol, high fat, nonalcoholic substances and pathological conditions through post-translational protein modifications

Mitochondria are critically important in providing cellular energy ATP as well as their involvement in anti-oxidant defense, fat oxidation, intermediary metabolism and cell death processes. It is well-established that mitochondrial functions are suppressed when living cells or organisms are exposed to potentially toxic agents including alcohol, high fat diets, smoking and certain drugs or in many pathophysiological states through increased levels of oxidative/nitrative stress. Under elevated nitroxidative stress, cellular macromolecules proteins, DNA, and lipids can undergo different oxidative modifications, leading to disruption of their normal, sometimes critical, physiological functions. Recent reports also indicated that many mitochondrial proteins are modified via various post-translation modifications (PTMs) and primarily inactivated. Because of the recently-emerging information, in this review, we specifically focus on the mechanisms and roles of five major PTMs (namely oxidation, nitration, phosphorylation, acetylation, and adduct formation with lipid-peroxides, reactive metabolites, or advanced glycation end products) in experimental models of alcoholic and nonalcoholic fatty liver disease as well as acute hepatic injury caused by toxic compounds. We also highlight the role of the ethanol-inducible cytochrome P450-2E1 (CYP2E1) in some of these PTM changes. Finally, we discuss translational research opportunities with natural and/or synthetic anti-oxidants, which can prevent or delay the onset of mitochondrial dysfunction, fat accumulation and tissue injury.