Glutamate-induced HT22 Cell Death Research Articles

Urinary metabolites of isorhynchophylline in rats and their neuroprotective activities in the HT22 cell assay

Isorhynchophylline is one of the major alkaloids from the Uncaria hook possessing the effects of lowered blood pressure, vasodilatation and protection against ischemia-induced neuronal damage. However, the metabolic pathway of isorhynchophylline has not been fully reported yet. In this paper, the metabolism of isorhynchophylline was investigated in rats. Five metabolites were isolated by using solvent extraction and repeated chromatographic methods, and identified by spectroscopic methods including UV, MS, NMR and CD experiments. Three new compounds were identified as 5-oxoisorhynchophyllic acid-22-O-β-D-glucuronide (M1), 17-O-demethyl-16,17-dihydro isorhynchophylline (M2) and 5-oxoisorhynchophyllic acid (M4) together with two known compounds isorhynchophylline (M0) and rhynchophylline (M3). Possible metabolic pathways of isorhynchophylline are proposed. Furthermore, the activity assay for all the metabolites showed that isorhynchophylline (M0) exhibited potent neuroprotective effects against glutamate-induced HT22 cell death. However, little or weak neuroprotective activities were observed for M1–M4. Our present study is important to further understand its metabolic fate and disposition in humans.

Alkaloids from the hook-bearing branch of Uncariarhynchophylla and their neuroprotective effects against glutamate-induced HT22 cell death

One new alkaloid, 4-geissoschizine N-oxide methyl ether (1), was isolated from the EtOH extract of the hook-bearing branch of Uncariarhynchophylla, together with 10 known alkaloids, 3-epi-geissoschizine methyl ether (2) isolated from U.rhynchophylla for the first time, geissoschizine methyl ether (3), 4-hirsuteine N-oxide (4), hirsuteine (5), hirsutine (6), 3α-dihydro-cadambine (7), 3β-isodihydro-cadambine (8), cadambine (9), strictosamide (10), and akuammigine (11). The structures were elucidated by spectroscopic methods including UV, ESI-QTOF MS, NMR, and circular dichroism experiments. Neuroprotective effects of 1–9 were investigated against 3 mM glutamate-induced HT22 cell death. The activity assay showed that 2, 3, 5, and 6 exhibited potent neuroprotective effects against glutamate-induced HT22 cell death. However, only weak neuroprotective activities were observed for 1, 4, 7, 8, and 9.

Andrographolide, a major component of Andrographis paniculata leaves, has the neuroprotective effects on glutamate-induced HT22 cell death

Andrographis paniculata (AP) is a traditional herbal medicine that is used for the treatment of inflammation-related disorders, dyspepsia, and diarrhea. Recently, the biological effects of AP on cancer, microbial infections, cardiovascular disease, and inflammation were reported. However, neuroprotective actions of AP during glutamate-induced oxidative stress have not been investigated. In this study, we isolated andrographolide (AG) as an active compound from the ethanolic extract of AP leaves, and evaluated its neuroprotective mechanisms using glutamate-treated HT22 mouse hippocampal neuronal cells. Five mM glutamate reduced cell survival significantly to 55.90 ± 2.16%. However, 5 μM AG restored cell viability to 102.19 ± 13.98%. AG decreased the early apoptosis by inhibiting Ca2+ influx, intracellular reactive oxygen species production, and lipid peroxidation. Moreover, AG regulated the levels of Bcl-2, Bid, Bax, and apoptosis-inducing factor. AG also inhibited the phosphorylation of mitogen activated protein kinases including p38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase.

Neuroprotective effect of the fermented Gumiganghwal-tang

Gumiganghwal-tang (GT) is a traditional herbal prescription widely used to treat inflammatory diseases in Asia. In this study, we evaluated neuroprotective effect and acetylcholinesterase (AChE) inhibitory activity of GT and compared with fermented GT (FGT). In order to better understand the neuroprotective mechanism, intracellular reactive oxygen species (ROS) production was investigated and high-performance liquid chromatography with diode-array detection (HPLC-DAD) analysis of contents of GT and FGT were conducted. As a result, FGT showed more potent protective effect against glutamate-induced HT22 cell death than GT and inhibited AChE activity. The neuroprotective effect of FGT is associated with inhibition of ROS production. The HPLC-DAD analytical results indicated that FGT contains higher content of bergapten and atractylenolide III than that of GT. In conclusion, FGT have neuroprotective effect and AChE inhibition, and may be useful source for treatment of neurodegenerative disease.

Selectivity, Cocrystal Structures, and Neuroprotective Properties of Leucettines, a Family of Protein Kinase Inhibitors Derived from the Marine Sponge Alkaloid Leucettamine B

DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) are implicated in the onset and development of Alzheimer's disease and Down syndrome. The marine sponge alkaloid leucettamine B was recently identified as an inhibitor of DYRKs/CLKs. Synthesis of analogues (leucettines) led to an optimized product, leucettine L41. Leucettines were cocrystallized with DYRK1A, DYRK2, CLK3, PIM1, and GSK-3β. The selectivity of L41 was studied by activity and interaction assays of recombinant kinases and affinity chromatography and competition affinity assays. These approaches revealed unexpected potential secondary targets such as CK2, SLK, and the lipid kinase PIKfyve/Vac14/Fig4. L41 displayed neuroprotective effects on glutamate-induced HT22 cell death. L41 also reduced amyloid precursor protein-induced cell death in cultured rat brain slices. The unusual multitarget selectivity of leucettines may account for their neuroprotective effects. This family of kinase inhibitors deserves further optimization as potential therapeutics against neurodegenerative diseases such as Alzheimer's disease.

Identification of tyrosine‐nitrated proteins in HT22 hippocampal cells during glutamate‐induced oxidative stress

Nitration of tyrosine residues in protein is a post-translational modification, which occurs under oxidative stress, and is associated with several neurodegenerative diseases. To understand the role of nitrated proteins in oxidative stress-induced cell death, we identified nitrated proteins and checked correlation of their nitration in glutamate-induced HT22 cell death. Nitrated proteins were detected by western blotting using an anti-nitrotyrosine antibody, extracted from matching reference 2-dimensional electrophoresis gels, and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Glutamate treatment induced apoptosis in HT22 cells, while reactive oxygen species (ROS) inhibitor or neuronal nitric oxide synthase (nNOS) inhibitor blocked glutamate-induced HT22 cell death. Nitration levels of 13 proteins were increased after glutamate stimulation; six of them were involved in regulation of energy production and two were related to apoptosis. The other nitrated proteins were associated with calcium signal modulation, ER dysfunction, or were of unknown function. The 13 tyrosine-nitrated proteins were detected in these glutamate-treated HT22 cells. Results demonstrated that cell death, ROS accumulation and nNOS expression were related to nitration of protein tyrosine in the glutamate-stimulated cells.

(Arylthio)cyclopentenones derivatives prevent glutamate-induced HT22 cell death through a PPARγ-dependent pathway

We show that cyclopentenone derivatives, which are synthetic analogs of cyclopentenone prostaglandins, are neuroprotective against glutamate-induced oxidative stress in HT22 cells. This effect was antagonized by a peroxisome proliferator-activated receptor-γ (PPARγ) antagonist, T0070907. Pull-down assays revealed that biotinylated (arylthio)cyclopentenone (GIF-0643-biotin) and other biotin-bound cyclopentenones bind to PPARγ. The results also indicate that the GIF-0643-biotin–PPARγ complex is localized to the nucleus. Moreover, retinoid X receptor-α (RXR) co-precipitated with the GIF-0643-biotin–PPARγ complex, indicating that (arylthio)cyclopentenone can activate PPARγ-RXR heterodimers. These findings suggest that (arylthio)cyclopentenone derivatives prevent excitotoxicity by modulating gene expression via activation of the PPARγ-RXR hetrodimer.

Activation of autophagy during glutamate-induced HT22 cell death

Recent evidence suggests that autophagy plays a role in oxidative injury-induced cell death. Here we examined whether glutamate-mediated oxidative toxicity induces autophagy in murine hippocampal HT22 cells and if autophagy induction affects the molecular events associated with cell death. Markers for autophagy induction including LC3 conversion, suppression of mTOR pathway, and GFP-LC3 dot formation were enhanced by glutamate treatment. By contrast, autophagy inhibition blocked glutamate-induced LC3 conversion and consequently reduced cell death. Activation of ERK1/2, a hallmark of glutamate-induced cytotoxicity, was also decreased by autophagy inhibition. Interestingly, autophagy inhibition also affected the expression of chaperones including Hsp60 and Hsp70, which are differentially regulated during HT22 cell death. Conversely, knock-down of Hsp60 greatly decreased LC3 conversion. Together these results suggest that glutamate-induced cytotoxicity involves autophagic cell death and chaperones may play a role in this process.

Proteomic analysis of glutamate‐induced toxicity in HT22 cells

In the present study, we have investigated the proteome changes associated with glutamate-induced HT22 cell death, a model system to study oxidative stress-mediated toxicity. Among a number of HT22 proteins exhibiting altered expression, several molecular chaperones demonstrated substantial changes. For example, the levels of Hsp90 and Hsp70 decreased as cell death progressed whereas that of Hsp60 increased dramatically. Interestingly, cytosolic Hsp60 increased more prominently than mitochondrial Hsp60. Concomitantly, the accumulation of poly-ubiquitylated proteins and differential regulation of the peptidase activities and the subunits of 26S proteasomes were observed in glutamate-treated HT22 cells. Our findings that the molecular chaperones and the ubiquitin-proteasome system undergo changes during glutamate-induced HT22 cell death may suggest the importance of a protein quality control system in oxidative damage-mediated toxicity.

Curcumin attenuates glutamate-induced HT22 cell death by suppressing MAP kinase signaling

Glutamate induces cell death by upsetting the cellular redox homeostasis, termed oxidative glutamate toxicity, in a mouse hippocampal cell line, HT22. Extracellular signal-regulated kinases (ERK) 1/2 are known key players in this process. Here we characterized the roles of both MAP kinases and cell cycle regulators in mediating oxidative glutamate toxicity and the neuroprotective mechanisms of curcumin in HT22 cells. c-Jun N-terminal kinase (JNK) and p38 kinase were activated during the glutamate-induced HT22 cell death, but at a later stage than ERK activation. Treatment with a JNK inhibitor, SP600125, or a p38 kinase inhibitor, SB203580, partly attenuated this cell death. Curcumin, a natural inhibitor of JNK signaling, protected the HT22 cells from glutamate-induced death at nanomolar concentrations more efficiently than SP600125. These doses of curcumin affected neither the level of intracellular glutathione nor the level of reactive oxygen species, but inactivated JNK and p38 significantly. Moreover, curcumin markedly upregulated a cell-cycle inhibitory protein, p21cip1, and downregulated cyclin D1 levels, which might help the cell death prevention. Our results suggest that curcumin has a neuroprotective effect against oxidative glutamate toxicity by inhibiting MAP kinase signaling and influencing cell-cycle regulation.

Testosterone increases neurotoxicity of glutamate in vitro and ischemia-reperfusion injury in an animal model.

Increasing evidence has demonstrated striking sex differences in the outcome of neurological injury. Whereas estrogens contribute to these differences by attenuating neurotoxicity and ischemia-reperfusion injury, the effects of testosterone are unclear. The present study was undertaken to determine the effects of testosterone on neuronal injury in both a cell-culture model and a rodent ischemia-reperfusion model. Glutamate-induced HT-22 cell-death model was used to evaluate the effects of testosterone on cell survival. Testosterone was shown to significantly increase the toxicity of glutamate at a 10 microM concentration, whereas 17beta-estradiol significantly attenuated the toxicity at the same concentration. In a rodent stroke model, ischemia-reperfusion injury was induced by temporal middle cerebral artery occlusion (MCAO) for 1 h and reperfusion for 24 h. To avoid the stress-related testosterone reduction, male rats were castrated and testosterone was replaced by testosterone pellet implantation. Testosterone pellets were removed at 1, 2, 4, or 6 h before MCAO to determine the duration of acute testosterone depletion effects on infarct volume. Ischemic lesion volume was significantly decreased from 239.6 +/- 25.9 mm(3) in control to 122.5 +/- 28.6 mm(3) when testosterone pellets were removed at 6 h before MCAO. Reduction of lesion volume was associated with amelioration of the hyperemia during reperfusion. Our in vitro and in vivo studies suggest that sex differences in response to brain injury are partly due to the consequence of damaging effects of testosterone.