Biomedical subjects

Chul-Sang Lee

Publications and source records attributed to Chul-Sang Lee.

7 recordsLinked to original sources

PubMed2005

Lentiviral transduction of murine oligodendrocytes in vivo.

Lentiviral vectors are used widely to direct efficient gene transfer in vivo. We examined cell-specific expression in adult murine white matter after stereotaxic microinjection of four lentiviral constructs. We synthesized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviruses with combinations of two promoters, cytomegalovirus (CMV) or myelin basic protein (MBP), and two reporter sequences, cytosolic enhanced green fluorescent protein (eGFP) or a plasma membrane-targeted eGFP (human lymphocyte-specific protein tyrosine kinase [Lck]-eGFP). For all constructs, intracerebral injections to lateral corpus callosum resulted in widespread GFP expression in forebrain white matter glial cells. Intense cellular GFP fluorescence was observed within 3 days after injection and lasted for at least 28 days. The CMV promoter directed GFP expression in multiple glial cell types, whereas the MBP promoter targeted GFP specifically to oligodendrocytes. Expression of the membrane-targeted Lck-eGFP construct distinctly labeled individual myelinating processes of oligodendrocytes. Lentiviral constructs expressing eGFP or Lck-eGFP under the MBP promoter provide excellent visualization of oligodendrocyte morphology in intact white matter, and may prove valuable for delivering additional genes of interest to oligodendrocytes in vivo.

Animals↗

PubMed2005

Epac1-mediated Rap1 activation is not required for the production of nitric oxide in BV2, murine microglial cells.

This study demonstrates that cyclic AMP (cAMP) production is induced by lipopolysaccharide (LPS) stimulation and activates two different pathways in murine BV2 microglial cells. Two principal effector proteins for cAMP are protein kinase A (PKA) and cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor. When cells were treated with various cAMP level modulators, nitric oxide (NO) production increased as the result of posttreatment with Type IV phosphodiesterase (PDE4) inhibitor, rolipram or dibutyryl-cAMP (dbcAMP), at 2 hr after LPS stimulation. Intracellular cAMP increased due to LPS stimulation and the cAMP modulators phosphorylate transcription factor CREB, which is enhanced in turn by posttreatment with dbcAMP. In contrast, the Epac-specific cAMP analog 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP) activates Rap1 in the BV2 cells, but does not induce PKA activation, as judged by CREB phosphorylation. NO production was enhanced by posttreatment with dbcAMP but not by treatment with 8CPT-2Me-cAMP. This suggests that LPS-stimulated NO production is mainly PKA-dependent and also that Epac1-mediated Rap1 activation is not required for the induction of NO production.

Analysis of Variance↗

PubMed2005

Neurotrophin and GDNF family ligands promote survival and alter excitotoxic vulnerability of neurons derived from murine embryonic stem cells.

Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4-/4+ protocol (Bain et al., Dev Biol 168:342-57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRalpha1, GFRalpha2, and ret was confirmed by immunocytochemistry. Exposure to 30-100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 microM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons.

Animals↗

PubMed2005

Transient decrease in F-actin may be necessary for translocation of proteins into dendritic spines.

It remains poorly understood as to how newly synthesized proteins that are required to act at specific synapses are translocated into only selected subsets of potentiated dendritic spines. Here, we report that F-actin, a major component of the skeletal structure of dendritic spines, may contribute to the regulation of synaptic specificity of protein translocation. We found that the stabilization of F-actin blocked the translocation of GFP-CaMKII and inhibited the diffusion of 3-kDa dextran into spines (in 2-3 weeks cultures). Neuronal activation in hippocampal slices and cultured neurons led to an increase in the activation (decrease in the phosphorylation) of the actin depolymerization factor, cofilin, and a decrease in F-actin. Furthermore, the induction of long-term potentiation by tetanic stimulation induced local transient depolymerization of F-actin both in vivo and in hippocampal slices (8-10 weeks), and this local F-actin depolymerization was blocked by APV, a N-methyl-D-aspartate (NMDA) receptor antagonist. These results suggest that F-actin may play a role in synaptic specificity by allowing protein translocation into only potentiated spines, gated through its depolymerization, which is probably triggered by the activation of NMDA receptors.

2-Amino-5-phosphonovalerate↗

PubMed2004

A proteasomal stress response: pre-treatment with proteasome inhibitors increases proteasome activity and reduces neuronal vulnerability to oxidative injury.

We report here that exposure to low concentrations of proteasome inhibitors (e.g. 10-100 nm MG-132, 0.1-3 nm epoxomicin or 10-30 nm clasto-lactacystin beta-lactone) resulted in an enhancement, rather than an inhibition, of proteasome activity in cultured neocortical neurons. Size-fractionation chromatography confirmed that the enhanced peptide cleavage activity was associated with proteasome-sized complexes. This sub toxic exposure reduced neuronal death caused by subsequent exposure to oxidative stress (100-200 microm H(2)O(2) for 30 min, 24-h exposure to 100 microm paraquat or 7.5 microm menadione), but did not alter vulnerability to excitotoxicity (5-min exposure to 30-100 microm NMDA or 24 exposure to 12 microm NMDA). Sub toxic proteasome inhibitor exposure caused an increase in levels of proteasome core subunit proteins and mRNAs, but not in levels of potentially cytoprotective heat shock proteins (hsp70, hsp90 and hsp40). The neuroprotective effects of proteasome inhibitor pre-treatment were blocked by coapplication of proteasome inhibitors during the oxidative insult. These findings support a model in which sublethal proteasome inhibition induces neurons to increase proteasome activity and promotes resistance to oxidative injury and suggests that enhancement of proteasome activity is a potential therapeutic target for diseases in which oxidative stress has been implicated.

Animals↗

PubMed2002

Retinoic acid response element in HOXA-7 regulatory region affects the rate, not the formation of anterior boundary expression.

Since it is known that a 307 bp fragment of the position specific regulatory element of human HOXA-7 contains two (DR3 and DR5) retinoic acid response elements (RAREs) at its 3' end, we constructed several deletion constructs containing different numbers of RAREs and examined their effects in vitro and in vivo. The 5' deletion constructs, BM112 and OM213, retaining both RAREs, were highly responsible (about 8 fold induction) for RA in F9 teratocarcinoma cells, versus NM307 (4-5 fold). The construct NS218, with both RAREs deleted but retaining the 5' sequences lost RA responsibility completely, whereas NR271, with one RARE(DR5) deleted retained a 50% inducibility (2.5 fold). In vivo transgenic analysis revealed that the constructs NM307 and NR271, but not OM213 nor BM112, directed the position specific expression pattern. Sequence analysis revealed that HOXA-7 enhancer sequences, including the RARE repeat sequences, were well conserved in human, mouse and chick. Part of the RAREs overlaps with the CDX1 binding site, and sequences of the DR3 RAREs were identical in this species. Two GAGA binding sites were also found to be strictly conserved. Because OM213, which had one GAGA site disrupted but retaining both RAREs, did not direct anterior boundary formation in transgenic mice, these results suggest the importance of the 5' 94 bp region, including the GAGA binding site, in anterior boundary formation and the involvement of the RARE in the rate of expression not in anterior boundary formation.

Animals↗

PubMedDate not supplied

Transplantation of embryonic stem cells overexpressing Bcl-2 promotes functional recovery after transient cerebral ischemia.

The study tested the hypothesis that transplantation of embryonic stem (ES) cells into rat cortex after a severe focal ischemia would promote structural repair and functional recovery. Overexpression of the human anti-apoptotic gene bcl-2 in ES cells was tested for increasing survival and differentiation of transplanted cells and promoting functional benefits. Mouse ES cells, pretreated with retinoic acid to induce differentiation down neural lineages, were transplanted into the post-infarct brain cavity of adult rats 7 days after 2-h occlusion of the middle cerebral artery (MCA). Over 1-8 weeks after transplantation, the lesion cavity filled with ES cell-derived cells that expressed markers for neurons, astrocytes, oligodendrocytes, and endothelial cells. ES cell-derived neurons exhibited dendrite outgrowth and formed a neuropil. ES cell-transplanted animals exhibited enhanced functional recovery on neurological and behavioral tests, compared to control animals injected with adult mouse cortical cells or vehicle. Furthermore, transplantation with ES cells overexpressing Bcl-2 further increased the survival of transplanted ES cells, neuronal differentiation, and functional outcome. This study supports that ES cell transplantation and gene modification may have values for enhancing recovery after stroke.

Animals↗