Dohoon Kim

Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins

Document S1. Supplemental Experimental Procedures, Supplemental References, Eight Figures, and Four TablesxDownload (.47 MB ) Document S1. Supplemental Experimental Procedures, Supplemental References, Eight Figures, and Four Tables

SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis

A progressive loss of neurons with age underlies a variety of debilitating neurological disorders, including Alzheimers disease (AD) and amyotrophic lateral sclerosis (ALS), yet few effective treatments are currently available. The SIR2 gene promotes longevity in a variety of organisms and may underlie the health benefits of caloric restriction, a diet that delays aging and neurodegeneration in mammals. Here, we report that a human homologue of SIR2, SIRT1, is upregulated in mouse models for AD, ALS and in primary neurons challenged with neurotoxic insults. In cell‐based models for AD/tauopathies and ALS, SIRT1 and resveratrol, a SIRT1‐activating molecule, both promote neuronal survival. In the inducible p25 transgenic mouse, a model of AD and tauopathies, resveratrol reduced neurodegeneration in the hippocampus, prevented learning impairment, and decreased the acetylation of the known SIRT1 substrates PGC‐1alpha and p53. Furthermore, injection of SIRT1 lentivirus in the hippocampus of p25 transgenic mice conferred significant protection against neurodegeneration. Thus, SIRT1 constitutes a unique molecular link between aging and human neurodegenerative disorders and provides a promising avenue for therapeutic intervention.

Functional genomics reveal that the serine synthesis pathway is essential in breast cancer

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation (1,2). RNAi-based loss of function screening has proven powerful for the identification of novel and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumor suppressor genes (3). Here, we developed a method for identifying novel cancer targets via negative selection RNAi screening in solid tumours. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumourigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of ER-negative breast cancers. PHGDH catalyzes the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have elevations in serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of alpha-ketoglutarate, another output of the pathway and a TCA cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH over-expression and demonstrate the utility of in vivo negative selection RNAi screens for finding potential anticancer targets.

A novel pathway regulates memory and plasticity via SIRT1 and miR-134

The NAD-dependent deacetylase Sir2 was initially identified as a mediator of replicative lifespan in budding yeast and was subsequently shown to modulate longevity in worms and flies. Its mammalian homologue, SIRT1, seems to have evolved complex systemic roles in cardiac function, DNA repair and genomic stability. Recent studies suggest a functional relevance of SIRT1 in normal brain physiology and neurological disorders. However, it is unknown if SIRT1 has a role in higher-order brain functions. We report that SIRT1 modulates synaptic plasticity and memory formation via a microRNA-mediated mechanism. Activation of SIRT1 enhances, whereas its loss-of-function impairs, synaptic plasticity. Surprisingly, these effects were mediated via post-transcriptional regulation of cAMP response binding protein (CREB) expression by a brain-specific microRNA, miR-134. SIRT1 normally functions to limit expression of miR-134 via a repressor complex containing the transcription factor YY1, and unchecked miR-134 expression following SIRT1 deficiency results in the downregulated expression of CREB and brain-derived neurotrophic factor (BDNF), thereby impairing synaptic plasticity. These findings demonstrate a new role for SIRT1 in cognition and a previously unknown microRNA-based mechanism by which SIRT1 regulates these processes. Furthermore, these results describe a separate branch of SIRT1 signalling, in which SIRT1 has a direct role in regulating normal brain function in a manner that is disparate from its cell survival functions, demonstrating its value as a potential therapeutic target for the treatment of central nervous system disorders.

Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production

The Akt/protein kinase B (PKB) serine/ threonine kinase is well known as an important mediator of many cell survival signaling pathways. Here, we demonstrate for the first time a major role of Akt/PKB in the cell invasion properties of the highly metastatic cell line HT1080. Using confocal microscopic analyses of live samples, we found Akt/PKB to be localized in the leading edge membrane area of migrating HT1080 cells. This localization was dependent on phosphoino‐sitide 3‐kinase and required the lipid binding ability of the phosphoinositide binding pleckstrin homology domain of Akt/PKB. We examined the possible function of Akt/PKB in HT1080 invasion. Surprisingly, Akt/ PKB potently promoted HT1080 invasion, by increasing cell motility and matrix metalloproteinase‐9 (MMP‐9) production, in a manner highly dependent on its kinase activity and membrane‐translocating ability. The increase in MMP‐9 production was mediated by activation of nuclear factor‐κB transcriptional activity by Akt/PKB. However, Akt/PKB did not affect the cell‐cell or cell‐matrix adhesion properties of HT1080. Our findings thus establish Akt/PKB as a major factor in the invasive abilities of cancer cells.

Hemangioblastic Derivatives from Human Induced Pluripotent Stem Cells Exhibit Limited Expansion and Early Senescence

Human induced pluripotent stem cells (hiPSC) have been shown to differentiate into a variety of replacement cell types. Detailed evaluation and comparison with their human embryonic stem cell (hESC) counterparts is critical for assessment of their therapeutic potential. Using established methods, we demonstrate here that hiPSCs are capable of generating hemangioblasts/blast cells (BCs), endothelial cells, and hematopoietic cells with phenotypic and morphologic characteristics similar to those derived from hESCs, but with a dramatic decreased efficiency. Furthermore, in distinct contrast with the hESC derivatives, functional differences were observed in BCs derived from hiPSCs, including significantly increased apoptosis, severely limited growth and expansion capability, and a substantially decreased hematopoietic colony‐forming capability. After further differentiation into erythroid cells, >1,000‐fold difference in expansion capability was observed in hiPSC‐BCs versus hESC‐BCs. Although endothelial cells derived from hiPSCs were capable of taking up acetylated low‐density lipoprotein and forming capillary‐vascular‐like structures on Matrigel, these cells also demonstrated early cellular senescence (most of the endothelial cells senesced after one passage). Similarly, retinal pigmented epithelium cells derived from hiPSCs began senescing in the first passage. Before clinical application, it will be necessary to determine the cause and extent of such abnormalities and whether they also occur in hiPSCs generated using different reprogramming methods. STEM CELLS 2010;28:704–712

Deregulation of HDAC1 by p25/Cdk5 in neurotoxicity

Aberrant cell-cycle activity and DNA damage are emerging as important pathological components in various neurodegenerative conditions. However, their underlying mechanisms are poorly understood. Here, we show that deregulation of histone deacetylase 1 (HDAC1) activity by p25/Cdk5 induces aberrant cell-cycle activity and double-strand DNA breaks leading to neurotoxicity. In a transgenic model for neurodegeneration, p25/Cdk5 activity elicited cell-cycle activity and double-strand DNA breaks that preceded neuronal death. Inhibition of HDAC1 activity by p25/Cdk5 was identified as an underlying mechanism for these events, and HDAC1 gain of function provided potent protection against DNA damage and neurotoxicity in cultured neurons and an in vivo model for ischemia. Our findings outline a pathological signaling pathway illustrating the importance of maintaining HDAC1 activity in the adult neuron. This pathway constitutes a molecular link between aberrant cell-cycle activity and DNA damage and is a potential target for therapeutics against diseases and conditions involving neuronal death.

Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease

Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell-based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.

p25/Cyclin-Dependent Kinase 5 Induces Production and Intraneuronal Accumulation of Amyloid β In Vivo

Aberrant processing of the amyloid precursor protein (APP) and the subsequent accumulation of amyloid β (Aβ) peptide has been widely established as a central event in Alzheimers disease (AD) pathogenesis. The sequential cleavage steps required for the generation of Aβ are well outlined; however, there is a relative dearth of knowledge pertaining to signaling pathways and molecular mechanisms that can modulate this process. Here, we demonstrate a novel role for p25/cyclin-dependent kinase 5 (Cdk5) in regulating APP processing, Aβ peptide generation, and intraneuronal Aβ accumulation in inducible p25 transgenic and compound PD–APP transgenic mouse models that demonstrate deregulated Cdk5 activity and a neurodegenerative phenotype. Induction of p25 resulted in enhanced forebrain Aβ levels before any evidence of neuropathology in these mice. Intracellular Aβ accumulated in perinuclear regions and distended axons within the forebrains of these mice. Evidence for modulations in axonal transport or β-site APP cleaving enzyme 1 protein levels and activity are presented as mechanisms that may account for the Aβ accumulation caused by p25/Cdk5 deregulation. Collectively, these findings delineate a novel pathological mechanism involving aberrant APP processing by p25/Cdk5 and have important implications in AD pathogenesis.

Defective regulation of autophagy upon leucine deprivation reveals a targetable liability of human melanoma cells in vitro and in vivo

Autophagy is of increasing interest as a target for cancer therapy. We find that leucine deprivation causes the caspase-dependent apoptotic death of melanoma cells because it fails to appropriately activate autophagy. Hyperactivation of the RAS-MEK pathway, which is common in melanoma, prevents leucine deprivation from inhibiting mTORC1, the main repressor of autophagy under nutrient-rich conditions. In an in vivo tumor xenograft model, the combination of a leucine-free diet and an autophagy inhibitor synergistically suppresses the growth of human melanoma tumors and triggers widespread apoptosis of the cancer cells. Together, our study represents proof of principle that anticancer effects can be obtained with a combination of autophagy inhibition and strategies to deprive tumors of leucine.