Neuronal HT22 Research Articles

Protective effects of berries and walnuts against the accelerated aging and age‐associated stress caused by irradiation in critical regions of rat brain

The occurrence of neurodegenerative disease substantially increases with age, which, in part, may be due to increased susceptibility to oxidative stress, inflammation and loss of autophagy (neuronal housekeeping). Polyphenols and ω‐fatty acids, abundant in berries (e.g., blueberry, strawberry, or acai fruit) and walnuts, have been shown to protect brain cells in culture and animals against oxidative stress/inflammation, with enhanced memory and cognitive function in animals. We have investigated whether feeding rats with blueberry‐ or strawberry‐supplemented diets, followed by irradiation with high energy and charge (HZE) particles, a model for accelerated aging, would elicit any protective effects in the brain. HZE irradiation disrupted key proteins in the hippocampus and striatum. Feeding animals with either berry diet, prior to irradiation, protected these brain regions against inflammation, oxidative stress and loss of autophagy. Moreover, autophagy activation was mediated by inhibiting the phosphorylation of mTOR and activating other proteins. Walnuts also elicited similar effects on the normal aging process when fed to aged animals. These molecular effects were further corroborated in vitro using BV2 microglia, HT22 neurons and E18 neuron‐astrocyte cultures. This study extends molecular evidences for the health‐promoting properties of berries and walnuts.Grant Funding Source: USDA intramural grant

Neuroprotective and anti-inflammatory effects of flavonoids isolated from Rhus verniciflua in neuronal HT22 and microglial BV2 cell lines

The neuroprotective and anti-inflammatory activities of the methanolic extract of Rhus verniciflua Stokes (Anacardiaceae) were investigated with mouse hippocampal and microglial cells. Bioactivity-guided isolation yielded 10 flavonoids including fustin (1), fisetin (2), sulfuretin (3), butein (4), butin (5), eriodictyol (6), morin hydrate (7), quercetin (8), kaempferol (9) and isoliquiritigenin (10). Among the isolated flavonoids, compounds 2–5 significantly protected the murine hippocampal HT22 cells against glutamate-induced neurotoxicity and attenuated reactive oxygen species (ROS) generations. In addition, these flavonoids significantly maintained antioxidative defense systems preserving the activities of superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GSH-Px) and the content of glutathione (GSH) decreased by glutamate insult. These compounds also showed significant inhibitory effects on LPS-induced nitric oxide (NO) production in BV2 cells. Especially, compound 4 dose-dependently suppressed the expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). These results suggest that these flavonoids possess therapeutic potentials as a multipotent agent against neurodegenerative diseases related to oxidative stress and pathological inflammatory responses.

Differentiation of HT22 neurons induces expression of NMDA receptor that mediates homocysteine cytotoxicity

Introduction: Neurotoxic homocysteine (Hcy) is thought to be an independent risk factor for neurodegenerative diseases, including Alzheimer’s disease. This study is to determine whether HT22 cells, a murine hippocampal neuronal model, can be used as an in vitro model, besides the primary neuronal cultures, to investigate the effects of Hcy.Materials and methods: MTS assay and Hoechst 33342/propidium iodide discrimination were used to assess the cell viability and cell death on undifferentiated and differentiated HT22 cells. Semi-quantitative reverse transcription polymerase chain reaction and western blot were used to determine the expression of N-methyl D-aspartate (NMDA) receptor.Results: We found that undifferentiated and differentiated HT22 cells responded to Hcy toxicity differentially, with the undifferentiated cells resistant while the differentiated cells sensitive. The underlying mechanism appeared to be the differential expression levels of NMDA glutamate receptor between the undifferentiated and differentiated cells. Similar to what have been observed in primary neuronal cultures, the Hcy toxicity in the differentiated HT22 cells was largely attenuated by NMDA receptor antagonists, MK-801 and memantine.Conclusion: These results suggest for the first time that the differentiation of HT22 cells could induce the expression of NMDA receptors, which lead to Hcy mediate concentration-dependent apoptosis-necrotic continuum of HT22 cell death. The differentiation status of the HT22 cells is important for modeling neurons in vitro, with the differentiated HT22 neurons resembling more characteristics of primary hippocampal neurons while the undifferentiated HT22 cells being proliferating neuronal precursor cells. The differentiated HT22 neurons can be used as a platform to study Hcy toxicity.

Glycogen synthase kinase 3β inhibitors protect hippocampal neurons from radiation-induced apoptosis by regulating MDM2-p53 pathway

Exposure of the brain to ionizing radiation can cause neurocognitive deficiencies. The pathophysiology of these neurological changes is complex and includes radiation-induced apoptosis in the subgranular zone of the hippocampus. We have recently found that inhibition of glycogen synthase kinase 3β (GSK-3β) resulted in significant protection from radiation-induced apoptosis in hippocampal neurons. The molecular mechanisms of this cytoprotection include abrogation of radiation-induced accumulation of p53. Here we show that pretreatment of irradiated HT-22 hippocampal-derived neurons with small molecule inhibitors of GSK-3β SB216763 or SB415286, or with GSK-3β-specific shRNA resulted in accumulation of the p53-specific E3 ubiquitin ligase MDM2. Knockdown of MDM2 using specific shRNA or chemical inhibition of MDM2-p53 interaction prevented the protective changes triggered by GSK-3β inhibition in irradiated HT-22 neurons and restored radiation cytotoxicity. We found that this could be due to regulation of apoptosis by subcellular localization and interaction of GSK-3β, p53 and MDM2. These data suggest that the mechanisms of radioprotection by GSK-3β inhibitors in hippocampal neurons involve regulation of MDM2-dependent p53 accumulation and interactions between GSK-3β, MDM2 and p53.

Abstract 2511: Glycogen synthase kinase 3 beta inhibitors protect hippocampal neurons from radiation-induced apoptosis by regulating MDM2/p53 pathway

Abstract Exposure of the brain to ionizing radiation can cause neurocognitive deficiencies. The pathophysiology of these neurological changes is complex and includes radiation-induced apoptosis in the subgranular zone of the hippocampus. We have recently found that inhibition of glycogen synthase kinase 3β (GSK-3β) resulted in significant protection from radiation-induced apoptosis in hippocampal neurons. The Molecular mechanisms of this cytoprotection include abrogation of radiation-induced accumulation of p53. Here we show that pretreatment of irradiated HT-22 neurons with small molecule inhibitors of GSK-3β SB216763 or SB415286, or with GSK-3β-specific shRNA resulted in accumulation of the p53-specific E3 ubiquitin ligase MDM2. Knockdown of MDM2 using specific shRNA or chemical inhibition of MDM2 prevented the protective changes triggered by GSK-3β inhibition in irradiated HT-22 and restored radiation cytotoxicity. We found that this could be due to regulation of apoptosis by subcellular localization and interaction of GSK-3β, p53 and MDM2. These data suggest that the mechanisms of radioprotection by GSK-3β inhibitors in hippocampal neurons involve regulation of MDM2-dependent p53 accumulation and interactions between GSK-3β, MDM2 and p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2511. doi:10.1158/1538-7445.AM2011-2511

Anti-neuroinflammatory and anti-oxidative activities of the ethanol extract from Citrus unshiu MARC.

Citrus peel extract has shown anti-inflammatory and anti-oxidative effects in several tissues. Therefore ，we investigated whether the ethanol extract from Fruit peel of Citrus unshiu MARC. suppresses brain insult-induced excessive microglial activation and neuronal oxidative stress. Releases of NO，the major inflammatory mediator in microglia，and iNOS mRNA expressions were markedly inhibited in a dose-dependent manner following treatment of the ethanol extract from Citrus unshiu MARC. in LPS-stimulated BV-2 microglia cells. In addition, the extract significantly suppressed LPS-induced mRNA expression of pro-inflammatory cytokine TNF-α IL-lβ and IL-6. On the other hand, the thanol extract exerted intracellular ROS scavenging activities in HT-22 neurons in a dose dependent manner (50-400 ug/ml)， suggesting the potent anti-oxidative stress effects in neurons. Taken together，these results suggest that Citrus unshiu MARC. peel extract has therapeutic potential against neuroinflammation and neurodegeneration via suppression of brain insult-induced microglial activation and neuronal oxidative stresses.

Serum deprivation induced autophagy and predominantly an AIF-dependent apoptosis in hippocampal HT22 neurons

Neuronal death induced by serum deprivation (SD) in HT22-cells was accompanied by a moderate activation of caspase-3, a prominent upregulation of AIF and its translocation into the nucleus. In addition protein levels of autophagy markers such as LC3 and beclin-1 were affected by SD. The ratio of LC3-II/LC3-I was significantly increased in serum deprived cultures. Furthermore, the addition of the pan-caspase inhibitor z-VAD(OMe)-FMK (zVAD) does not protect HT22-cells from SD-induced neurodegeneration. However, addition of the autophagy inhibitors such as 3-methyladenine (3-MA) or bafilomycin A1 (BafA1) provided a potentiation of cell death induced by SD. z-VAD and 3-MA in combination were not only ineffective in rescuing cells from the damaging effects of SD, but seem likely to act in synergy to potentiate slightly SD-induced cell death. The results of the current study suggest that SD induced predominantly caspase-independent apoptosis in hippocampal HT22 cells and that inhibition of autophagy is rather deleterious than protective.

Basal Levels of eIF2α Phosphorylation Determine Cellular Antioxidant Status by Regulating ATF4 and xCT Expression

eIF2alpha is part of a multimeric complex that regulates cap-dependent translation. Phosphorylation of eIF2alpha (phospho-eIF2alpha) is induced by various forms of cell stress, resulting in changes to the proteome of the cell with two diametrically opposed consequences, adaptation to stress or initiation of programmed cell death. In contrast to the robust eIF2alpha phosphorylation seen in response to acute insults, less is known about the functional role of basal levels of eIF2alpha phosphorylation. Here we show that mouse embryonic fibroblasts expressing a nonphosphorylatable eIF2alpha have enhanced sensitivity to diverse toxic insults, including amyloid beta-(1-42) peptide (Abeta), a key factor in the pathogenesis of Alzheimer disease. This correlates with impaired glutathione metabolism because of down-regulation of the light chain, xCT, of the cystine/glutamate antiporter system X(-)(c). The mechanistic link between the absence of phospho-eIF2alpha and xCT expression is nuclear factor ATF4. Consistent with these findings, long term activation of the phospho-eIF2alpha/ATF4/xCT signaling module by the specific eIF2alpha phosphatase inhibitor, salubrinal, induces resistance against oxidative glutamate toxicity in the hippocampal cell line HT22 and primary cortical neurons. Furthermore, in PC12 cells selected for resistance against Abeta, increased activity of the phospho-eIF2alpha/ATF4/xCT module contributes to the resistant phenotype. In wild-type PC12 cells, activation of this module by salubrinal ameliorates the response to Abeta. Furthermore, in human brains, ATF4 and phospho-eIF2alpha levels are tightly correlated and up-regulated in Alzheimer disease, most probably representing an adaptive response against disease-related cellular stress rather than a correlate of neurodegeneration.

Oxygen-sensitive reset of hypoxia-inducible factor transactivation response: Prolyl hydroxylases tune the biological normoxic set point

Cellular O 2 sensing enables physiological adjustments to variations in tissue pO 2. Under basal conditions, cells are adjusted to an O 2 environment biologically read as normoxia. Any sharp departure from that state of normoxia triggers O 2-sensitive biological responses. The stabilization of hypoxia-inducible factor (HIF) signifies a robust biological readout of hypoxia. In the presence of sufficient O 2, HIF is hydroxylated and degraded. HIF prolyl hydroxylation is catalyzed by prolyl hydroxylase isoenzymes PHD1, 2, and 3. Using HT22 neurons stably transfected with a HIF reporter construct, we tested a novel hypothesis postulating that biological cells are capable of resetting their normoxic set point by O 2-sensitive changes in PHD expression. Results of this study show that the pO 2 of the mouse brain cortex was 35 mm Hg or 5% O 2. Exposure of HT22, adjusted to growing in 20% O 2, to 5% O 2 resulted in HIF-driven transcription. However, cells adjusted to growing in 5% O 2 did not report hypoxia. Cells adjusted to growing in 30% O 2 reported hypoxia when acutely exposed to room air culture conditions. When grown under high O 2 conditions, cells reset their normoxic set point upward by down-regulating the expression of PHD1–3. When grown under low O 2 conditions, cells reset their normoxic set point downward by inducing the expression of PHD1–3. Exposure of mice in vivo to a hypoxic 10% O 2 environment lowered blood as well as brain pO 2. Such hypoxic exposure induced PHD1–3. Exposure of mice to a hyperoxic 50% O 2 ambience repressed the expression of PHD1–3, indicating that O 2-sensitive regulation of PHD expression is effective in the brain in vivo. siRNA dependent knockdown of PHD expression revealed that O 2-sensitive regulation of PHD may contribute to tuning the normoxic set point in biological cells.

Role of Protein Phosphatases in Estrogen-Mediated Neuroprotection

The signaling pathways that mediate neurodegeneration are complex and involve a balance between phosphorylation and dephosphorylation of signaling and structural proteins. We have shown previously that 17beta-estradiol and its analogs are potent neuroprotectants. The purpose of this study was to delineate the role of protein phosphatases (PPs) in estrogen neuroprotection against oxidative stress and excitotoxicity. HT-22 cells, C6-glioma cells, and primary rat cortical neurons were exposed to the nonspecific serine/threonine protein phosphatase inhibitors okadaic acid and calyculin A at various concentrations in the presence or absence of 17beta-estradiol and/or glutamate. Okadaic acid and calyculin A caused a dose-dependent decrease in cell viability in HT-22, C6-glioma, and primary rat cortical neurons. 17beta-Estradiol did not show protection against neurotoxic concentrations of either okadaic acid or calyculin A in these cells. In the absence of these serine/threonine protein phosphatase inhibitors, 17beta-estradiol attenuated glutamate toxicity. However, in the presence of effective concentrations of these protein phosphatase inhibitors, 17beta-estradiol protection against glutamate toxicity was lost. Furthermore, glutamate treatment in HT-22 cells and primary rat cortical neurons caused a 50% decrease in levels of PP1, PP2A, and PP2B protein, whereas coadministration of 17beta-estradiol with glutamate prevented the decrease in PP1, PP2A, and PP2B levels. These results suggest that 17beta-estradiol may protect cells against glutamate-induced oxidative stress and excitotoxicity by activating a combination of protein phosphatases.

Neuroprotective Properties of Afobazol in Vitro

The effects of a novel selective anxiolytic afobazol on survival of HT-22 neurons were studied in the model of oxidative stress and glutamate toxicity. In both models, the neuroprotective effect of afobazol was established.

Differential effects of glucocorticoids and gonadal steroids on glutathione levels in neuronal and glial cell systems

The aim of the present study was to investigate the short- and long-term effects of glucocorticoids [corticosterone (CORT), dexamethasone (DEX), 6-methylprednisolone (6-MP)] and gonadal steroids [17beta-estradiol (E(2)), progesterone (PROG), testosterone (TEST)] on the levels of the antioxidant glutathione (GSH) in different cell systems of the CNS (neuronal hippocampal HT22 cells, primary hippocampal and neocortical brain cells, and C(6) glioma cells). In HT22 cells, steroids exerted mainly long-term effects. Significant increases of GSH levels were detectable after a 24 hr treatment with 10(-7) M of DEX (122% +/- 5%), 6-MP (208% +/- 32%), E(2) (134% +/- 10%), and TEST (155% +/- 17%). A significant decrease occurred after incubation with PROG for 24 hr (79% +/- 9%). In primary hippocampal cultures, a 24 hr treatment with DEX (140% +/- 8%), E(2) (123% +/- 6%), and PROG (118% +/- 5%) led to significant increases of the GSH levels, whereas, in neocortical primary cultures, only an incubation with E(2) increased GSH (149% +/- 8%). In C(6) cells, hormone treatment led to both significant short-term (1 hr: CORT 114% +/- 5%, DEX 90% +/- 3%, E(2) 88% +/- 3%; 3 hr: DEX 115% +/- 5%, E(2) 122% +/- 6%, TEST 78% +/- 4%) and significant long-term (24 hr: CORT 74% +/- 4%, 6-MP 84% +/- 5%, E(2) 115% +/- 6%, PROG 91% +/- 4%, TEST 116% +/- 5%) effects. In summary, we were able to demonstrate differential effects of steroids on GSH levels in different cellular CNS models, showing an important influence of steroids and especially E(2) on antioxidative cellular functions in neuronal and glial cells.