• Biomolecules & Therapeutics
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• v.20 no.3
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• pp.306-312
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• 2012

Resveratrol (trans-3,5,4'-trihydroxystilbene) has received considerable attention recently for the potential neuroprotective effects in neurodegenerative disorders where heme oxygenase-1 (HO-1) and sirtuin 1 (SIRT1) represent promising therapeutic targets. Resveratrol has been known to increase HO-1 expression and SIRT1 activity. In this study, the effects of resveratrol and trans-3,5,4'-trimethoxystilbene (TMS), a resveratrol derivative, on cytotoxicity caused by glutamate-induced oxidative stress, HO-1 expression, and SIRT1 activation have been investigated by using murine hippocampal HT22 cells, which have been widely used as an in vitro model for investigating glutamate-induced neurotoxicity. Resveratrol protected HT22 neuronal cells from glutamate-induced cytotoxicity and increased HO-1 expression as well as SIRT1 activity in a concentration-dependent manner. Cytoprotection afforded by resveratrol was partially reversed by the specific inhibition of HO-1 expression by HO-1 small interfering RNA and the nonspecific blockage of HO-1 activity by tin protoporphyrin IX, but not by SIRT1 inhibitors. Surprisingly, TMS, a resveratrol derivative with methoxyl groups in lieu of the hydroxyl groups, and trans-stilbene, a non-hydroxylated analog, failed to protect HT22 cells from glutamate-induced cytotoxicity and to increase HO-1 expression and SIRT1 activity. Taken together, our findings suggest that the cytoprotective effect of resveratrol was at least in part associated with HO-1 expression but not with SIRT1 activation and, importantly, that the presence of hydroxyl groups on the benzene rings of resveratrol appears to be necessary for cytoprotection against glutamate-induced oxidative stress, HO-1 expression, and SIRT1 activation in HT22 neuronal cells.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.195-202
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• 2018

Hyperglycemia is one of the major risk factors for stroke. Hyperglycemia can lead to a more extensive infarct volume, aggravate neuronal damage after cerebral ischemia. ${\alpha}$-Synuclein is especially abundant in neuronal tissue, where it underlies the etiopathology of several neurodegenerative diseases. This study investigated whether hyperglycemic conditions regulate the expression of ${\alpha}$-synuclein in middle cerebral artery occlusion (MCAO)-induced cerebral ischemic injury. Male Sprague-Dawley rats were treated with streptozotocin (40 mg/kg) via intraperitoneal injection to induce hyperglycemic conditions. MCAO were performed four weeks after streptozotocin injection to induce focal cerebral ischemia, and cerebral cortex tissues were obtained 24 hours after MCAO. We confirmed that MCAO induced neurological functional deficits and cerebral infarction, and these changes were more extensive in diabetic animals compared to non-diabetic animals. Moreover, we identified a decrease in ${\alpha}$-synuclein after MCAO injury. Diabetic animals showed a more serious decrease in ${\alpha}$-synuclein than non-diabetic animals. Western blot and reverse-transcription PCR analyses confirmed more extensive decreases in ${\alpha}$-synuclein expression in MCAO-injured animals with diabetic condition than these of non-diabetic animals. It is accepted that ${\alpha}$-synuclein modulates neuronal cell death and exerts a neuroprotective effect. Thus, the results of this study suggest that hyperglycemic conditions cause more serious brain damage in ischemic brain injuries by decreasing ${\alpha}$-synuclein expression.

• Journal of Ginseng Research
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• v.47 no.2
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• pp.274-282
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• 2023

Background: Ginsenoside compound K (CK) stimulated activation of the PI3K-Akt signaling is one of the major mechanisms in promoting cell survival after stroke. However, the underlying mediators remain poorly understood. This study aimed to explore the docking protein of ginsenoside CK mediating the neuroprotective effects. Materials and methods: Molecular docking, surface plasmon resonance, and cellular thermal shift assay were performed to explore ginsenoside CK interacting proteins. Neuroscreen-1 cells and middle cerebral artery occlusion (MCAO) model in rats were utilized as in-vitro and in-vivo models. Results: Ginsenoside CK interacted with recombinant human PTP1B protein and impaired its tyrosine phosphatase activity. Pathway and process enrichment analysis confirmed the involvement of PTP1B and its interacting proteins in PI3K-Akt signaling pathway. PTP1B overexpression reduced the tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) after oxygen-glucose deprivation/reoxygenation (OGD/R) in neuroscreen-1 cells. These regulations were confirmed in the ipsilateral ischemic hemisphere of the rat brains after MCAO/R. Ginsenoside CK treatment reversed these alterations and attenuated neuronal apoptosis. Conclusion: Ginsenoside CK binds to PTP1B with a high affinity and inhibits PTP1B-mediated IRS1 tyrosine dephosphorylation. This novel mechanism helps explain the role of ginsenoside CK in activating the neuronal protective PI3K-Akt signaling pathway after ischemia-reperfusion injury.

• 한국약용작물학회:학술대회논문집
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• 2011.09a
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• pp.31-45
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• 2011

The amyloid ${\beta}$-peptide ($A{\beta}$), which originates from the proteolytic cleavage of amyloid precursor protein (APP), plays a central role in the pathogenesis of Alzheimer's disease (AD). Mounting evidence indicates that different species of $A{\beta}$, such as $A{\beta}$ oligomers and fibrils, may contribute to AD pathogenesis via distinct mechanisms at different stages of the disease. Importantly, elevation and accumulation of soluble $A{\beta}$ oligomers closely correlate with cognitive decline and/or disease progression in animal models of AD. In agreement with these studies, oligomers of $A{\beta}$ have been shown to directly affect synaptic plasticity, a neuronal process that is known to be essential for memory formation. Our previous studies showed that $A{\beta}$ induces the breakdown of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a phospholipid that regulates key aspects of neuronal function. PI(4,5)P2 breakdown was found to be a key step toward synaptic and memory dysfunction in a mouse model of AD. To this end, we seek to identify small molecules that could elevate the levels of PI(4,5)P2 and subsequently block $A{\beta}$ oligomer-induced breakdown of PI(4,5)P2 and synaptic dysfunction.. We found that (20S)Rg3, an active triterpene glycoside from heat-processed ginseng, serves as an agonist for phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha), which is a lipid kinase that mediates a rate-limiting step in PI(4,5)P2 synthesis. Consequently, (20S)Rg3 stimulates PI(4,5)P2 synthesis by directly stimulating the activity of PI4KIIalpha. Interestingly, treatment of a mouse model of AD with (20S)Rg3 leads to reversal of memory deficits. Our data suggest that the PI(4,5)P2-promoting effects of (20S)Rg3 may help mitigate the cognitive symptoms associated with AD.

• Biomolecules & Therapeutics
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• v.31 no.1
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• pp.116-126
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• 2023

Mainly due to the slanted focus on the mechanism and regulation of neuronal aging, research on astrocyte aging and its modulation during brain aging is scarce. In this study, we established aged astrocyte culture model by long-term culturing. Cellular senescence was confirmed through SA-β-gal staining as well as through the examination of morphological, molecular, and functional markers. RNA sequencing and functional analysis of astrocytes were performed to further investigate the detailed characteristics of the aged astrocyte model. Along with aged phenotypes, decreased astrocytic proliferation, migration, mitochondrial energetic function and support for neuronal survival and differentiation has been observed in aged astrocytes. In addition, increased expression of cytokines and chemokine-related factors including plasminogen activator inhibitor -1 (PAI-1) was observed in aged astrocytes. Using the RNA sequencing results, we searched potential drugs that can normalize the dysregulated gene expression pattern observed in long-term cultured aged astrocytes. Among several candidates, minoxidil, a pyrimidine-derived anti-hypertensive and anti-pattern hair loss drug, normalized the increased number of SA-β-gal positive cells and nuclear size in aged astrocytes. In addition, minoxidil restored up-regulated activity of PAI-1 and increased mitochondrial superoxide production in aged astrocytes. We concluded that long term culture of astrocytes can be used as a reliable model for the study of astrocyte senescence and minoxidil can be a plausible candidate for the regulation of brain aging.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.4
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• pp.253-257
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• 2013

This study examined the mechanism of action of a local anesthetic, lidocaine HCl. Energy transfer between the surface fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid, and the hydrophobic fluorescent probe, 1,3-di(1-pyrenyl) propane, was used to determine the effect of lidocaine HCl on the thickness (D) of the synaptosomal plasma membrane vesicles (SPMV) isolated from the bovine cerebral cortex, and liposomes of the total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. The thickness (D) of the intact SPMV, SPMVTL and SPMVPL were $1.044{\pm}0.008$, $0.914{\pm}0.005$ and $0.890{\pm}0.003$ (arbitrary units, n=5) at $37^{\circ}C$ (pH 7.4), respectively. Lidocaine HCl decreased the thickness of the neuronal and model membrane lipid bilayers in a dose-dependent manner with a significant decrease in the thickness, even at 0.1 mM. The decreasing effect of lidocaine HCl on the membrane thickness might be responsible for some, but not all of its anesthetic action.

• International Journal of Oral Biology
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• v.35 no.4
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• pp.159-167
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• 2010

To provide a basis for studying the pharmacological actions of tetracaine HCl, we analyzed the membrane activities of this local anesthetic. The n-(9-anthroyloxy) stearic and palmitic acid (n-AS) probes (n = 2, 6, 9, 12 and 16) have been used previously to examine fluorescence polarization gradients. These probes can report the environment at a graded series of depths from the surface to the center of the membrane bilayer structure. In a dosedependent manner, tetracaine HCl decreased the anisotropies of 6-AS, 9-AS, 12-AS and 16-AP in the hydrocarbon interior of synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV), and liposomes derived from total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. However, this compound increased the anisotropy of 2-AS at the membrane interface. The magnitude of the membrane rotational mobility reflects the carbon atom numbers of the phospholipids comprising SPMV, SPMVTL and SPMVPL and was in the order of the 16, 12, 9, 6, and 2 positions of the aliphatic chains. The sensitivity of the effects of tetracaine HCl on the rotational mobility of the hydrocarbon interior or surface region was dependent on the carbon atom numbers in the descending order 16-AP, 12-AS, 9-AS, 6-AS and 2-AS and on whether neuronal or model membranes were involved in the descending order SPMV, SPMVPL and SPMVTL.

• Development and Reproduction
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• v.12 no.1
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• pp.1-8
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• 2008

Specific protocols to increase the differentiation of neuronal cells from embryonic stem (ES) cells have been well established, such as retinoic acid induction and lineage selection of neuronal cells. For the neuropathological studies, ES-derived neurons (ES neurons) must show normal physiological characteristics related to cell death and survival and should be maintained in vitro for a sufficient time to show insults-specific cell death without spontaneous death. When mouse ES cells were plated onto astrocytes monolayer after retinoic acid induction, most ES cells differentiated into neuronal cells, which were confirmed by the presence of specific neuronal markers, and the cultures were viable for at least four weeks. When these cultures were examined for vulnerability to glutamate excitotoxicity, ES neurons were vulnerable to excitotoxic insults mediated by agonist-specific receptors. The vulnerability to excitotoxic death increased with developmental age of ES neurons in vitro. Specific receptors for Neurotrophin and GDNF family ligands were present in ES neurons. GDNF and NT-3 could modulate the survival and excitotoxic vulnerability of ES neurons. The vulnerability and resistance to toxic insults, which are essential requirements of model culture systems for neuropathological studies, make ES neurons to a useful model culture system. Especially ES cell are highly amenable to genetic modification unlikely to primary neuronal cells, which will give us a chance to answer more complicated neurophysiological questions. Recently there was an outstanding attempt to explore the cellular toxicity using human ES cells (Schrattenholz & Klemm, 2007) and it suggested that ES cells could be a new model system for neurophysiological studies soon and go further a large-scale screening system for pharmacological compounds in the future.

• Animal cells and systems
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• v.13 no.3
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• pp.267-274
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• 2009

The dissociative culture technique of Aplysia neuron is one of the key methods that have been used for studies of cellular and molecular mechanisms of neuronal functioning. However, despite the advantages this method offers as an experimental model, its technical efficiency has had room for improvement. In this study, we examined certain putative factors that might affect the culture quality. The effects of neuronal damage induced by physical injuries, heat shock, and surface protein degradation were evaluated along with the correlation between the culture quality and animal behavior. As a result, we found that physical injury can be a critical factor that affects culture quality, whereas the heat shock and surface protein degradation had negligible effect on it. In addition, we discovered that siphon retraction time was not a good measurement for healthy neurons. Based on these findings, we suggest here an improved method in which the degree of physical injury is reduced by means of multiple protease treatment.

• Nutrition Research and Practice
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• v.4 no.6
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• pp.455-461
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• 2010

The tumor microenvironment, particularly sufficient nutrition and oxygen supply, is important for tumor cell survival. Nutrition deprivation causes cancer cell death. Since apoptosis is a major mechanism of neuronal loss, we explored neuronal apoptosis in various microenvironment conditions employing neuroblastoma (NB) cells. To investigate the effects of tumor malignancy and differentiation on apoptosis, the cells were exposed to poor microenvironments characterized as serum-free, low-glucose, and hypoxia. Incubation of the cells in serum-free and low-glucose environments significantly increased apoptosis in less malignant and more differentiated N-type IMR32 cells, whereas more malignant and less differentiated I-type BE(2)C cells were not affected by those treatments. In contrast, hypoxia (1 % $O_2$) did not affect apoptosis despite cell malignancy. It is suggested that DLK1 constitutes an important stem cell pathway for regulating self-renewal, clonogenicity, and tumorigenicity. This raises questions about the role of DLK1 in the cellular resistance of cancer cells under poor microenvironments, which cancer cells normally encounter. In the present study, DLK1 overexpression resulted in marked protection from apoptosis induced by nutrient deprivation. This in vitro model demonstrated that increasing severity of nutrition deprivation and knock-down of DLK1 caused greater apoptotic death, which could be a useful strategy for targeted therapies in fighting NB as well as for evaluating how nutrient deprived cells respond to therapeutic manipulation.