Jong Seong Ha

Glutamate-induced oxidative stress, but not cell death, is largely dependent upon extracellular calcium in mouse neuronal HT22 cells.

Elucidating the relationship of glutamate-induced Ca2+ flux and oxidative death of neuronal cells may be of great relevance for neurodegenerative diseases in human beings. Mouse hippocampal HT22 cells provide a model system to study this relationship at the molecular level. Here we show that stimulation of HT22 cells with 5 mM glutamate is cytotoxic. Glutamate-induced cytotoxicity was associated with the generation of reactive oxygen species (ROS) and activation of the death executioner caspases 1 and 3. Treatment of HT22 cells with the calcium chelator, EGTA, and the calcium channel blocker, CoCl2, revealed that glutamate-induced cell death was dependent, in part, on glutamate-induced Ca2+ influx from extracellular stores. However, activation of caspases 1 and 3 and death of HT22 cells were also observed when Ca2+ was lacking in the extracellular milieu and ROS production abrogated. These findings led us to conclude that glutamate-induced death of mouse HT22 cells utilizes a complex mechanism that relies only in part on Ca2+ influx and ROS production. Additional studies are warranted to evaluate glutamate-induced death mechanisms that operate independently of Ca2+ influx and generation of ROS.

Nox4-dependent H2O2 production contributes to chronic glutamate toxicity in primary cortical neurons

Reactive oxygen species (ROS) can trigger neuronal cell death and has been implicated in a variety of neurodegenerative diseases as well as brain ischemia. Here, we demonstrate that chronic (but not acute) glutamate toxicity in primary cortical neuronal cultures is associated with hydrogen peroxide (H(2)O(2)) accumulation in the culture medium and that neurotoxicity can be eliminated by external catalase treatment. Neuronal cultures in Ca(2+)-free medium or treated with BAPTA showed reduced glutamate-induced H(2)O(2) generation, indicating that H(2)O(2) generation is Ca(2+)-dependent. Pharmacological and genetic approaches revealed that NADPH oxidase plays a role in glutamate-induced H(2)O(2) generation and that activation of NMDA and AMPA receptors is involved in this H(2)O(2) generation. The Nox4 siRNA reduced NMDA-induced H(2)O(2) production by 54% and cytotoxicity in parallel, suggesting that Nox4-containing NADPH oxidase functions NMDA receptor-mediated H(2)O(2) production resulting in neurotoxicity. These findings suggest that the modulation of NADPH oxidase can be used as a new therapeutic strategy for glutamate-induced neuronal diseases.

Extracellular hydrogen peroxide contributes to oxidative glutamate toxicity

Oxidative glutamate toxicity is characterized by the inhibition of cystine uptake, the depletion of intracellular glutathione, and increased levels of intracellular reactive oxygen species, factors that lead to neuronal injury. We found that the presence of extracellular catalase protected cultured neuronal cells, such as HT22, SH-SY5Y and PC12 cells, from glutamate-induced cytotoxicity. Extracellular hydrogen peroxide (H₂O₂) accumulated in a time- and concentration-dependent manner in HT22 cells during prolonged exposure to glutamate. To investigate the involvement of NADPH oxidase in glutamate-induced H₂O₂ generation, we used small interference RNA (siRNA). Knockdown of Nox2 and Nox4 expression reduced H₂O₂ accumulation and increased cell survival. siRNA specific for Nox4 reduced the production of H₂O₂ by ~74% compared with control siRNA. Furthermore, H₂O₂ accumulation was also suppressed by U0126, a MEK/ERK inhibitor, in a concentration-dependent manner. These results suggest that glutamate triggers the Nox-dependent generation of extracellular H₂O₂ via ERK1/2 activation, which contributes to oxidative glutamate toxicity.

Chronic glutamate toxicity in mouse cortical neuron culture.

Two pathways for glutamate toxicity have been described, receptor-mediated excitotoxicity and non-receptor mediated oxidative glutamate toxicity. Here, we show that two distinct forms of receptor-mediated primary cortical neuronal death exist, chronic and acute glutamate toxicity, and that these depend on exposure time. In vitro, neuronal sensitivity to chronic glutamate exposure increased as neurons matured and the initial plating medium contributed as well. In immature neurons, high concentrations of glutamate induced neuronal death. The chronic glutamate toxicity was independent of neuronal density, whereas increased density potentiated acute glutamate toxicity. Activation of ionotropic glutamate receptors (iGluRs) contributed to induction of chronic and acute glutamate toxicity at similar rates at DIV14. Inactivation of the metabotropic glutamate receptors (mGluRs) by AIDA increased neuronal sensitivity to chronic glutamate exposure but not to acute exposure. Neuronal death by acute toxicity was much faster than by chronic toxicity in which activation of mGluRs was involved. These results suggest that acute glutamate toxicity is quite different from chronic toxicity, in which activation of mGluRs is associated with resistance to glutamate toxicity.

Self-assembled mirror DNA nanostructures for tumor-specific delivery of anticancer drugs.

Nanoparticle delivery systems have been extensively investigated for targeted delivery of anticancer drugs over the past decades. However, it is still a great challenge to overcome the drawbacks of conventional nanoparticle systems such as liposomes and micelles. Various novel nanomaterials consist of natural polymers are proposed to enhance the therapeutic efficacy of anticancer drugs. Among them, deoxyribonucleic acid (DNA) has received much attention as an emerging material for preparation of self-assembled nanostructures with precise control of size and shape for tailored uses. In this study, self-assembled mirror DNA tetrahedron nanostructures is developed for tumor-specific delivery of anticancer drugs. l-DNA, a mirror form of natural d-DNA, is utilized for resolving a poor serum stability of natural d-DNA. The mirror DNA nanostructures show identical thermodynamic properties to that of natural d-DNA, while possessing far enhanced serum stability. This unique characteristic results in a significant effect on the pharmacokinetics and biodistribution of DNA nanostructures. It is demonstrated that the mirror DNA nanostructures can deliver anticancer drugs selectively to tumors with enhanced cellular and tissue penetration. Furthermore, the mirror DNA nanostructures show greater anticancer effects as compared to that of conventional PEGylated liposomes. Our new approach provides an alternative strategy for tumor-specific delivery of anticancer drugs and highlights the promising potential of the mirror DNA nanostructures as a novel drug delivery platform.

PI3K-ERK1/2 activation contributes to extracellular H2O2 generation in amyloid β toxicity.

Amyloid β peptide (Aβ) induces hydrogen peroxide (H2O2) and superoxide generation, leading to neuronal death. Many studies have shown the involvement of NADPH oxidase, but the isotype-specific role was not assessed. Moreover, the activation status of phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) 1/2 is unclear in extracellular H2O2 generation. In this paper, we showed that Aβ1-42 induced extracellular H2O2 generation and the resulting cytotoxicity in a concentration-dependent manner. Nox2- and Nox4-specific siRNAs suppressed H2O2 and superoxide generation. LY294002 and U0126, inhibitors of PI3K and ERK1/2, respectively, reduced H2O2 generation in concentration-dependent manners. Furthermore, PI3K activation is responsible for ERK1/2 phosphorylation. An additional increase in H2O2 generation and corresponding cytotoxicity was observed after treatment with Aβ1-42 and glutamate. These results suggest that Aβ1-42 enhances the neuronal vulnerability to oxidative injury in Alzheimers disease (AD) by increasing H2O2 generation.

Overcoming doxorubicin resistance of cancer cells by Cas9-mediated gene disruption

In this study, Cas9 system was employed to down-regulate mdr1 gene for overcoming multidrug resistance of cancer cells. Disruption of the MDR1 gene was achieved by delivery of the Cas9-sgRNA plasmid or the Cas9-sgRNA ribonucleoprotein complex using a conventional gene transfection agent and protein transduction domain (PTD). Doxorubicin showed considerable cytotoxicity to the drug-resistant breast cancer cells pre-treated with the RNA-guided endonuclease (RGEN) systems, whereas virtually non-toxic to the untreated cells. The potency of drug was enhanced in the cells treated with the protein-RNA complex as well as in those treated with plasmids, suggesting that mutation of the mdr1 gene by intracellular delivery of Cas9-sgRNA complex using proper protein delivery platforms could recover the drug susceptibility. Therefore, Cas9-mediated disruption of the drug resistance-related gene can be considered as a promising way to overcome multidrug resistance in cancer cells.

PI3Kγ contributes to MEK1/2 activation in oxidative glutamate toxicity via PDK1

The role of phosphoinositide 3‐kinase (PI3K) in oxidative glutamate toxicity is not clear. Here, we investigate its role in HT22 mouse hippocampal cells and primary cortical neuronal cultures, showing that inhibitors of PI3K, LY294002, and wortmannin suppress extracellular hydrogen peroxide (H2O2) generation and increase cell survival during glutamate toxicity in HT22 cells. The mitogen‐activated protein kinase kinase (MEK) inhibitor U0126 also reduced glutamate‐induced H2O2 generation and inhibited phosphorylation of extracellular signal‐regulated kinase (ERK) 1/2. LY294002 was seen to abolish phosphorylation of both ERK1/2 and Akt. A small interfering RNA (siRNA) study showed that PI3Kβ and PI3Kγ, rather than PI3Kα and PI3Kδ, contribute to glutamate‐induced H2O2 generation and cell death. PI3Kγ knockdown also inhibited glutamate‐induced ERK1/2 phosphorylation, whereas transfection with the constitutively active form of human PI3Kγ (PI3Kγ‐CAAX) triggered MEK1/2 and ERK1/2 phosphorylation and H2O2 generation without glutamate exposure. This H2O2 generation was reduced by inhibition of MEK. Transfection with kinase‐dead 3‐phosphoinositide‐dependent protein kinase 1 (PDK1‐KD) reduced glutamate‐induced ERK1/2 phosphorylation and H2O2 generation. Accordingly, cotransfection of cells with PDK1‐KD and PI3Kγ‐CAAX suppressed PI3Kγ‐CAAX‐triggered ERK1/2 phosphorylation and H2O2 generation. These results suggest that activation of PI3Kγ induces ERK1/2 phosphorylation, leading to extracellular H2O2 generation via PDK1 in oxidative glutamate toxicity.

Poly-sgRNA/siRNA ribonucleoprotein nanoparticles for targeted gene disruption

ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)‐associated protein‐9 nuclease (Cas9) can be used for the specific disruption of a target gene to permanently suppress the expression of the protein encoded by the target gene. Efficient delivery of the system to an intracellular target site should be achieved to utilize the tremendous potential of the genome‐editing tool in biomedical applications such as the knock‐out of disease‐related genes and the correction of defect genes. Here, we devise polymeric CRISPR/Cas9 system based on poly‐ribonucleoprotein (RNP) nanoparticles consisting of polymeric sgRNA, siRNA, and Cas9 endonuclease in order to improve the delivery efficiency. When delivered by cationic lipids, the RNP nanoparticles built with chimeric poly‐sgRNA/siRNA sequences generate multiple sgRNA‐Cas9 RNP complexes upon the Dicer‐mediated digestion of the siRNA parts, leading to more efficient disruption of the target gene in cells and animal models, compared with the monomeric sgRNA‐Cas9 RNP complex.

Astrocytic phospholipase A2 contributes to neuronal glutamate toxicity.

The role of astrocytes in glutamate toxicity has been controversial. Here, we show that astrocytes in neuron-astrocyte co-cultures increased neuronal sensitivity to chronic glutamate exposure but not to acute exposure. Enhanced neuronal toxicity by chronic exposure was dependent on astrocyte cell numbers. A reduced generation of extracellular H2O2 induced by glutamate was observed in co-cultures. Further, neuronal glutamate toxicity was not suppressed by NADPH oxidase (Nox) inhibitors, catalase or Nox4 knockdown in co-cultures, whereas these compounds effectively reduced the toxicity in pure neuron cultures. Instead, the intracellular scavenger of reactive oxygen species, N-acetylcysteine (NAC), reduced neuronal cytotoxicity in co-cultures, whereas catalase worked in pure neuron cultures. Lipoxygenase (LOX) inhibitors attenuated neuronal glutamate toxicity in co-cultures but not in pure neuron cultures. Neuronal 5-LOX activity was increased only in co-cultures, whereas 12-LOX activity was increased in both types of cultures. The cyclooxygenase (COX) inhibitors, indomethacin and NS-398, and the phospholipase A2 (PLA2) inhibitors, LY311727 and MAFP, more effectively reduced neuronal glutamate toxicity in co-cultures than in pure neuron cultures. However, in co-cultures, pre-treating neurons and astrocytes with the same inhibitors generated opposite results. COX inhibitors suppressed neuronal glutamate toxicity in pre-treated neurons rather than astrocytes, whereas PLA2 inhibitors reduced the toxicity in pre-treated astrocytes rather than neurons. Gene-specific knockdown of PLA2 confirmed these results. Knockdown of cPLA2α and/or sPLA2-V in astrocytes rather than in neurons more effectively reduced glutamate toxicity in co-cultures. These findings suggest that astrocytic PLA2 activity increases neuronal sensitivity to chronic glutamate exposure in neuron-astrocyte co-cultures.