Activity-dependent synaptic localization of processing bodies and their role in dendritic structural plasticity.

In neurons, transport of a subset of mRNAs to subcellular regions and their translation has a role in synaptic plasticity. Recent studies have suggested a control mechanism of this local translation through mRNA compartmentalization or degradation. Here we report that processing bodies (P-bodies), which are involved in mRNA degradation or storage, are transported to dendrites by conventional kinesin (KIF5A) as a motor protein. Neuronal activation induced by depolarization increased the colocalization of P-bodies with PSD-95 in dendrites. This neuronal activity increased the release of Nd1 and Arp2 mRNA from the P-bodies and, consequently, reversed the decrease of F-actin (induced by overexpression of Dcp1a) in the dendrites. Our data suggest that the activity-induced redistribution of P-bodies and mRNA release from P-bodies might have a role in synaptic structural plasticity by altering levels of mRNAs that are involved in the dynamics of the actin cytoskeleton in dendrites.

Functional in vivo and in vitro effects of 20q11.21 genetic aberrations on hPSC differentiation.

Human pluripotent stem cells (hPSCs) have promising therapeutic applications due to their infinite capacity for self-renewal and pluripotency. Genomic stability is imperative for the clinical use of hPSCs; however, copy number variation (CNV), especially recurrent CNV at 20q11.21, may contribute genomic instability of hPSCs. Furthermore, the effects of CNVs in hPSCs at the whole-transcriptome scale are poorly understood. This study aimed to examine the functional in vivo and in vitro effects of frequently detected CNVs at 20q11.21 during early-stage differentiation of hPSCs. Comprehensive transcriptome profiling of abnormal hPSCs revealed that the differential gene expression patterns had a negative effect on differentiation potential. Transcriptional heterogeneity identified by single-cell RNA sequencing (scRNA-seq) of embryoid bodies from two different isogenic lines of hPSCs revealed alterations in differentiated cell distributions compared with that of normal cells. RNA-seq analysis of 22 teratomas identified several differentially expressed lineage-specific markers in hPSCs with CNVs, consistent with the histological results of the altered ecto/meso/endodermal ratio due to CNVs. Our results suggest that CNV amplification contributes to cell proliferation, apoptosis, and cell fate specification. This work shows the functional consequences of recurrent genetic abnormalities and thereby provides evidence to support the development of cell-based applications.

Generation of a NESTIN-EGFP reporter human induced pluripotent stem cell line, KSCBi005-A-1, using CRISPR/Cas9 nuclease.

NESTIN, an intermediate filament, is a neuroectodermal marker involved in induced pluripotent stem cell (iPSC) differentiation toward neural lineages. Here, we introduced an EGFP reporter into the C-terminus of NESTIN in KSCBi005-A hiPSCs through homologous recombination using CRISPR/Cas9 nuclease. The successfully edited line was confirmed by sequencing and had a normal karyotype. It expressed EGFP upon induction of neural differentiation and exhibited potential for differentiation into three germ layers. KSCBi005-A-1 cells could be used to monitor the expression of NESTIN in differentiated cell types. This cell line is available at the National Stem Cell Bank, Korea National Institute of Health.

Generation of a PDX1-EGFP reporter human induced pluripotent stem cell line, KSCBi005-A-3, using the CRISPR/Cas9 system.

PDX1 plays a crucial role in the development and maintenance of ß-cells and directly regulates pancreatic ß-cell-specific transcription factors by binding to the insulin gene. Here, we introduced an EGFP reporter into the C-terminus of PDX1 in KSCBi005-A human induced pluripotent stem cells through homologous recombination using CRISPR/Cas9 nuclease. The cells had a normal karyotype, expressed several pluripotency markers, and maintained their differentiation potential. KSCBi005-A-3 cells can be used to monitor PDX1 expression in live cells during ß-cell differentiation; the cell line has been registered at the National Stem Cell Bank, Korea National Institute of Health.

Generation of a human induced pluripotent stem cell line, KSCBi003-A, from human adipose tissue-derived mesenchymal stem cells using a chromosomal integration-free system.

We generated a human induced pluripotent stem cell (hiPSC) line, KSCBi003-A, from adipose tissue-derived mesenchymal stem cells (Ad-MSCs) using a Sendai virus-based gene delivery system. We confirmed that the KSCBi003-A has a normal karyotype and short tandem repeat (STR)-based identities that match the parent cells. We also confirmed that the cell line expresses pluripotent stem cell markers such as Nanog, OCT4, SSEA-4, TRA-1-60, and TRA-1-81. We also analyzed that the KSCBi003-A has an ability to differentiate three germ layers (ectoderm, mesoderm, endoderm). This cell line is registered and available at the National Stem Cell Bank, Korea National Institute of Health.

Down-regulation of p21-activated serine/threonine kinase 1 is involved in loss of mesencephalic dopamine neurons.

BACKGROUND: Although the roles of p21-activated serine/threonine kinase 1 (PAK1) have been reported in some neurodegenerative diseases, details regarding neurodegeneration are still limited. Hence, we tried to determine the role of PAK1 and molecular mechanisms of neuronal death involved in neurodegeneration. RESULTS: Expression of a dominant-negative form of PAK1 (PAK1(H83,86L, K229R), PAK1-DN) decreased the cell viability and increased cell death induced by oxidative stress. Indeed, oxidative stress decreased the phosphorylation of PAK1 in neuroblastoma cells, cultured dopamine (DA) neurons, or rat midbrains. PAK1-DN reduced the level of Bcl-2 protein, through an ubiquitin/proteasome-dependent mechanism. The level of Bcl-2 may be regulated by PAK1-ERK signaling and/or PAK1, directly. Conversely, expression of an active form of PAK1 (PAK1(T423E), PAK1-CA) could recover both loss of DA neurons in the substantia nigra (SN) and behavioral defects in a 6-OHDA-induced hemiparkinsonian rat model. CONCLUSIONS: Our data suggest that the oxidative stress-induced down-regulation of PAK1 activity could be involved in the loss of mesencephalic DA neurons through modulation of neuronal death, suggesting a novel role of PAK1 as a molecular determinant and mechanisms in the pathogenesis of Parkinson's disease.

Structure-Activity Relationship of Indole-Tethered Pyrimidine Derivatives that Concurrently Inhibit Epidermal Growth Factor Receptor and Other Angiokinases.

Antiangiogenic agents have been widely investigated in combination with standard chemotherapy or targeted cancer agents for better management of advanced cancers. Therapeutic agents that concurrently inhibit epidermal growth factor receptor and other angiokinases could be useful alternatives to combination therapies for epidermal growth factor receptor-dependent cancers. Here, we report the synthesis of an indole derivative of pazopanib using a bioisosteric replacement strategy, which was designated MKP101. MKP101 inhibited not only the epidermal growth factor receptor with an IC50 value of 43 nM but also inhibited angiokinases as potently as pazopanib. In addition, MKP101 effectively inhibited vascular endothelial growth factor-induced endothelial proliferation, tube formation, migration of human umbilical vein endothelial cells and proliferation of HCC827, an epidermal growth factor receptor-addicted cancer cell line. A docking model of MKP101 and the kinase domain of the epidermal growth factor receptor was generated to predict its binding mode, and validated by synthesizing and evaluating MKP101 derivatives. Additionally, a study of structure-activity relationships of indolylamino or indolyloxy pyrimidine analogues derived from MKP101 demonstrated that selectivity for epidermal growth factor receptor and other angiokinases, especially vascular endothelial growth factor receptor 2 depends on the position of substituents on pyrimidine and the type of link between pyrimidine and the indole moiety. We believe that this study could provide a basis for developing angiokinase inhibitors having high affinity for the epidermal growth factor receptor, from the pyrimidine scaffold.