Generation of TGFBI knockout ABCG2+/ABCB5+ double-positive limbal epithelial stem cells by CRISPR/Cas9-mediated genome editing.

Corneal dystrophy is an autosomal dominant disorder caused by mutations of the transforming growth factor ß-induced (TGFBI) gene on chromosome 5q31.8. This disease is therefore ideally suited for gene therapy using genome-editing technology. Here, we isolated human limbal epithelial stem cells (ABCG2+/ABCB5+ double-positive LESCs) and established a TGFBI knockout using RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. An LESC clone generated with a single-guide RNA (sgRNA) targeting exon 4 of the TGFBI gene was sequenced in order to identify potential genomic insertions and deletions near the Cas9/sgRNA-target sites. A detailed analysis of the differences between wild type LESCs and the single LESC clone modified by the TGFBI-targeting sgRNA revealed two distinct mutations, an 8 bp deletion and a 14 bp deletion flanked by a single point mutation. These mutations each lead to a frameshift missense mutation and generate premature stop codons downstream in exon 4. To validate the TGFBI knockout LESC clone, we used single cell culture to isolate four individual sub-clones, each of which was found to possess both mutations present in the parent clone, indicating that the population is homogenous. Furthermore, we confirmed that TGFBI protein expression is abolished in the TGFBI knockout LESC clone using western blot analysis. Collectively, our results suggest that genome editing of TGFBI in LESCs by CRISPR/Cas9 may be useful strategy to treat corneal dystrophy.

Application of in utero electroporation of G-protein coupled receptor (GPCR) genes, for subcellular localization of hardly identifiable GPCR in mouse cerebral cortex.

Lysophosphatidic acid (LPA) is a lipid growth factor that exerts diverse biological effects through its cognate receptors (LPA1-LPA6). LPA1, which is predominantly expressed in the brain, plays a pivotal role in brain development. However, the role of LPA1 in neuronal migration has not yet been fully elucidated. Here, we delivered LPA1 to mouse cerebral cortex using in utero electroporation. We demonstrated that neuronal migration in the cerebral cortex was not affected by the overexpression of LPA1. Moreover, these results can be applied to the identification of the localization of LPA1. The subcellular localization of LPA1 was endogenously present in the perinuclear area, and overexpressed LPA1 was located in the plasma membrane. Furthermore, LPA1 in developing mouse cerebral cortex was mainly expressed in the ventricular zone and the cortical plate. In summary, the overexpression of LPA1 did not affect neuronal migration, and the protein expression of LPA1 was mainly located in the ventricular zone and cortical plate within the developing mouse cerebral cortex. These studies have provided information on the role of LPA1 in brain development and on the technical advantages of in utero electroporation.

Lysophosphatidic acid activates ß-catenin/T cell factor signaling, which contributes to the suppression of apoptosis in H19-7 cells.

Lysophosphatidic acid (LPA) is a lipid growth factor that regulates diverse cell functions, including cell proliferation, survival and apoptosis. LPA has been demonstrated to be involved in the regulation of cortical neurogenesis by increasing the survival of neural precursors. Previously, we reported that LPA stimulated the inactivation of glycogen synthase kinase 3 (GSK3) via the G protein-coupled LPA1 and LPA2 receptors, by which apoptosis is suppressed in H19-7 cells [an embryonic hippocampal progenitor cell (HPC) line]. Increasing numbers of studies have demonstrated that certain G protein-coupled receptors activate ß-catenin/T cell factor (TCF) signaling independently of Wnt, which is involved in cell fate determination, cell proliferation and cell survival. To determine whether LPA activates ß-catenin-mediated transcriptional activation pathways and whether ß-catenin/TCF signaling is involved in neurogenesis by controlling the survival of neural precursors, ß-catenin/TCF signaling cascades induced by LPA were investigated in the HPCs. Activation of ß-catenin/TCF signaling was determined by the nuclear translocation of ß-catenin and the transcriptional activation of a TCF reporter gene. The activation of ß-catenin/TCF signaling was blocked by pertussis toxin (PTX) and a protein kinase C (PKC) inhibitor. The expression of a constitutively active mutated form of GSK3ß activated ß-catenin/TCF signaling to comparable levels to those induced by LPA, and protected against apoptosis in differentiating H19-7 cells. These results showed that LPA activates ß-catenin/TCF signaling in a PTX- and PKC-dependent manner, which contributes to LPA-induced cell survival in the HPCs. Activation of ß-catenin/TCF signaling by LPA may be involved in neurogenesis by controlling the survival of neural precursors.

Human group V secretory phospholipase A2 is associated with lipid rafts and internalized in a flotillin­dependent pathway.

The mechanisms of secretory phospholipase A2 (sPLA2) action are not understood clearly. Previously, it was suggested that sPLA2s are internalized into cells for the targeting of sPLA2 to intracellular action sites. However, the mechanisms for sPLA2 internalization remain to be identified. The present study demonstrated for the first time that human group V sPLA2 (hVPLA2) is associated with lipid rafts and is internalized in a flotillin­dependent pathway. The lipid raft association was probed by cholesterol­sensitive enrichment of hVPLA2 in low­density fractions and co­patching of ganglioside GM1 rafts through cross­linking of hVPLA2 in HEK293 and CHO cells. The hVPLA2 associated with lipid rafts was shown to be internalized into HEK293 cells at a relatively rapid rate (t1/2 =16 min) and this internalization was inhibited by the knockdown of flotillin­1, but not by chlorpromazine, an inhibitor of clathrin­mediated endocytosis. Moreover, internalized hVPLA2 was shown to be colocalized extensively with flotillin­1 in a punctate structure, but not caveolin­1. These data revealed that the internalization of hVPLA2 is mediated by flotillin­1. Attenuation of arachidonic acid release from plasma membrane through the association of hVPLA2 with lipid rafts suggested that this association with lipid rafts may be important in protecting mammalian cells from excessive degradation of plasma membrane and trafficking hVPLA2 into intracellular targets.

Antiproliferative and cytotoxic effects of resveratrol in mitochondria-mediated apoptosis in rat b103 neuroblastoma cells.

Resveratrol, a natural compound, has been shown to possess anti-cancer, anti-aging, anti-inflammatory, anti-microbial, and neuroprotective activities. In this study, we examined the antiproliferative and cytotoxicity properties of resveratrol in Rat B103 neuroblastoma cells; although it's molecular mechanisms for the biological effects are not fully defined. Here, we examined the cellular cytotoxicity of resveratrol by cell viability assay, antiproliferation by BrdU assay, DNA fragmentation by DNA ladder assay, activation of caspases and Bcl-2 family proteins were detected by western blot analyses. The results of our investigation suggest that resveratrol increased cellular cytotoxicity of Rat B103 neuroblastoma cells in a dose-and time-dependent manner with IC(50) of 17.86 µM at 48 h. On the other hand, incubation of neuroblastoma cells with resveratrol resulted in S-phase cell cycle arrests which dose-dependently and significantly reduced BrdU positive cells through the downregulation of cyclin D1 protein. In addition, resveratrol dose-dependently and significantly downregulated the expression of anti-apoptotic protein includes Bcl-2, Bcl-xL and Mcl-1 and also activates cleavage caspase-9 and-3 via the downregulation of procaspase-9 and -3 in a dose-dependent manner which indicates that involvement of intrinsic mitochondria-mediated apoptotic pathway. In conclusion, resveratrol increases cellular cytotoxicity and inhibits the proliferation of B103 neuroblastoma cells by inducing mitochondria-mediated intrinsic caspase dependent pathway which suggests this natural compound could be used as therapeutic purposes for neuroblastoma malignancies.

Lysophosphatidic acid induces neurite retraction in differentiated neuroblastoma cells via GSK-3ß activation.

Lysophosphatidic acid (LPA) is a lipid growth factor that exerts diverse biological effects, including rapid neurite retraction and cell migration. Alterations in cell morphology, including neurite retraction, in neurodegenerative disorders such as Alzheimer's disease involve hyperphosphorylation of the cytoskeletal protein tau. Since LPA has been shown to induce neurite retraction in various cultured neural cells and the detailed underlying molecular mechanisms have not yet been elucidated, we investigated whether LPA induced neurite retraction through taumediated signaling pathways in differentiated neuroblastoma cells. When Neuro2a cells differentiated with retinoic acid (RA) were exposed to LPA, cells exhibited neurite retraction in a time-dependent manner. The retraction of neurites was accompanied by the phosphorylation of tau. The LPA-induced neurite retraction and tau phosphorylation in differentiated Neuro2a cells were significantly abolished by the glycogen synthase kinase-3ß (GSK-3ß) inhibitor lithium chloride. Interestingly, the LPA-stimulated tau phosphorylation and neurite retraction were markedly prevented by the administration of H89, an inhibitor of both cyclic-AMP dependent protein kinase (PKA) and cyclic-AMP response element-binding protein (CREB). Transfection of the dominant-negative CREBs, K-CREB and A-CREB, failed to prevent LPA-induced tau phosphorylation and neurite retraction in differentiated Neuro2a cells. Taken together, these results suggest that GSK-3ß and PKA, rather than CREB, play important roles in tau phosphorylation and neurite retraction in LPA-stimulated differentiated Neuro2a cells.

Lysophosphatidylcholine-induced apoptosis in H19-7 hippocampal progenitor cells is enhanced by the upregulation of Fas Ligand.

Lysophospholipids regulate a wide array of biological processes including apoptosis and neutrophil migration. Fas/Apo-1 and its ligand (FasL) participate in neuronal cell apoptosis causing various neurological diseases. Here, we use hippocampal neuroprogenitor cells to investigate how lysophosphatidylcholine (LPC) induces apoptosis in H19-7 hippocampal progenitor cells via Fas/Fas ligand-mediated apoptotic signaling pathway. Exposed cells with LPC presented on apoptotic morphology, positive TUNEL staining, and DNA fragmentation. We found that the expression of FasL was increased after LPC treatment. Furthermore, LPC-induced H19-7 cell apoptosis was decreased by agonistic anti-FasL antibody. In addition to promotion of caspase cascade activity by LPC, the administration of the caspase inhibitor, DEVD-fmk, prevented H19-7 cell apoptosis. LPC also increased the activation of nuclear factor-kappaB (NF-kappaB), which in turn, significantly increased FasL mRNA level. The increase in FasL mRNA level by NF-kappaB transfection was significantly decreased in the presence of IkappaB-SR, a super-repressor of IkappaB. Taken together, these results demonstrate that LPC has the ability to induce apoptosis in H19-7 cells through the upregulation of FasL expression via NF-kappaB activation.