Bokyung Kim

Mitochondrial ROS govern the LPS-induced pro-inflammatory response in microglia cells by regulating MAPK and NF-κB pathways.

Activation of microglia cells in the brain contributes to neurodegenerative processes promoted by many neurotoxic factors such as pro-inflammatory cytokines and nitric oxide (NO). Reactive oxygen species (ROS) actively affect microglia-associated neurodegenerative diseases through their role as pro-inflammatory molecules and modulators of pro-inflammatory processes. Although the ROS which involved in microglia activation are thought to be generated primarily by NADPH oxidase (NOX) and involved in the immune response, mitochondrial ROS have also been proposed as important regulators of the inflammatory response in the innate immune system. However, the role of mitochondrial ROS in microglial activation has yet to be fully elucidated. In this study, we demonstrate that inhibition of mitochondrial ROS by treatment with Mito-TEMPO effectively suppressed the level of mitochondrial and intracellular ROS. Mito-TEMPO treatment also significantly prevented LPS-induced increase in the TNF-α, IL-1β, IL-6, iNOS and Cox-2 in BV-2 and primary microglia cells. Furthermore, LPS-induced suppression of mitochondrial ROS generation not only affected LPS-stimulated activation of MAPKs, including ERK, JNK, and p38, but also regulated IκB activation and NF-κB nuclear localization. These results indicate that mitochondria constitute a major source of ROS generation in LPS-mediated activated microglia cells. Additionally, suppression of LPS-induced mitochondrial ROS plays a role in modulating the production of pro-inflammatory mediators by preventing MAPK and NF-κB activation in microglia cells. Our findings suggest that a potential strategy in the development of therapy for inflammation-associated degenerative neurological diseases involves targeting the regulation of mitochondrial ROS in microglial cells.

Loss of mitofusin 2 links beta-amyloid-mediated mitochondrial fragmentation and Cdk5-induced oxidative stress in neuron cells.

Mitochondrial dysfunction is implicated in age‐related degenerative disorders such as Alzheimers disease (AD). Maintenance of mitochondrial dynamics is essential for regulating mitochondrial function. Aβ oligomers (AβOs), the typical cause of AD, lead to mitochondrial dysfunction and neuronal loss. AβOs have been shown to induce mitochondrial fragmentation, and their inhibition suppresses mitochondrial dysfunction and neuronal cell death. Oxidative stress is one of the earliest hallmarks of AD. Cyclin‐dependent kinase 5 (Cdk5) may cause oxidative stress by disrupting the antioxidant system, including Prx2. Cdk5 is also regarded as a modulator of mitochondrial fission; however, a precise mechanistic link between Cdk5 and mitochondrial dynamics is lacking. We estimated mitochondrial morphology and alterations in mitochondrial morphology‐related proteins in Neuro‐2a (N2a) cells stably expressing the Swedish mutation of amyloid precursor protein (APP), which is known to increase AβO production. We demonstrated that mitochondrial fragmentation by AβOs accompanies reduced mitofusin 1 and 2 (Mfn1/2) levels. Interestingly, the Cdk5 pathway, including phosphorylation of the Prx2‐related oxidative stress, has been shown to regulate Mfn1 and Mfn2 levels. Furthermore, Mfn2, but not Mfn1, over‐expression significantly inhibits the AβO‐mediated cell death pathway. Therefore, these results indicate that AβO‐mediated oxidative stress triggers mitochondrial fragmentation via decreased Mfn2 expression by activating Cdk5‐induced Prx2 phosphorylation.

Iron overload triggers mitochondrial fragmentation via calcineurin-sensitive signals in HT-22 hippocampal neuron cells.

The accumulation of iron in neurons has been proposed to contribute to the pathology of numerous neurodegenerative diseases, such as Alzheimers disease and Parkinsons disease. However, insufficient research has been conducted on the precise mechanism underlying iron toxicity in neurons. In this study, we investigated mitochondrial dynamics in hippocampal HT-22 neurons exposed to ferric ammonium citrate (FAC) as a model of iron overload and neurodegeneration. Incubation with 150 μM FAC for 48 h resulted in decreased cell viability and apoptotic death in HT-22 cells. The FAC-induced iron overload triggered mitochondrial fragmentation, which was accompanied by Drp1(Ser637) dephosphorylation. Iron chelation with deferoxamine prevented the FAC-induced mitochondrial fragmentation and apoptotic cell death by inhibiting Drp1(Ser637) dephosphorylation. In addition, a S637D mutation of Drp1, which resulted in a phosphorylation-mimetic form of Drp1 at Ser637, protected against the FAC-induced mitochondrial fragmentation and neuronal apoptosis. FK506 and cyclosporine A, inhibitors of calcineurin activation, determined that calcineurin was associated with the iron-induced changes in mitochondrial morphology and the phosphorylation levels of Drp1. These results indicate that the FAC-induced dephosphorylation of Drp1-dependent mitochondrial fragmentation was rescued by the inhibition of calcineurin activation. Therefore, these findings suggest that calcineurin-mediated phosphorylation of Drp1(Ser637) acts as a key regulator of neuronal cell loss by modulating mitochondrial dynamics in iron-induced toxicity. These results may contribute to the development of novel therapies for treatment of neurodegenerative disorders related to iron toxicity.

Colorectal cancer-derived tumor spheroids retain the characteristics of original tumors

Primary cultures of cancer cells are useful for developing personalized medicine. In this study, we characterized three lines of three-dimensional (3D) tumor spheroids established directly from tumor tissues of patients with colorectal cancers (CRCs). Each line mainly included EpCAM-positive cells and cells expressing putative cancer stem cell markers such as CD133, CD44, CD24, ALDH1, and LGR5. These characteristic stem cell markers remained identically for months in vitro. Short tandem repeat genotyping suggested that genetic fingerprints of these tumor spheroids were similar to those of the original tumor tissues from which they were derived. Mutational analysis showed that each line had the same mutation profile for APC, KRAS, MLH1, serine-threonine kinase 11, and TP53 as its parental tumor tissue. One line harboring an activating KRAS mutation was resistant to cetuximab while the remaining two lines harboring wild-type KRAS showed different responses to cetuximab. Immunohistochemical analysis showed that xenograft tumors derived from these lines retained the histopathological and mutational patterns of their parental tumors. Collectively, these results clearly showed that 3D tumor spheroids directly generated from tumor tissues of patients with CRCs preserved the characteristics of their parental tumor tissues and could be used for developing personalized medicines for CRCs.

peroxiredoxin 5 prevents amyloid-beta oligomer-induced neuronal cell death by inhibiting Erk–drp1-mediated mitochondrial fragmentation

Alzheimers disease (AD), a neurodegenerative disorder, is caused by amyloid-beta oligomers (AβOs). AβOs induce cell death by triggering oxidative stress and mitochondrial dysfunction. A recent study showed that AβO-induced oxidative stress is associated with extracellular signal-regulated kinase (ERK)-dynamin related protein 1 (Drp1)-mediated mitochondrial fission. Reactive oxygen species (ROS) are regulated by antioxidant enzymes, especially peroxiredoxins (Prxs) that scavenge H2O2. These enzymes inhibit neuronal cell death induced by various neurotoxic reagents. However, it is unclear whether Prx5, which is specifically expressed in neuronal cells, protects these cells from AβO-induced damage. In this study, we found that Prx5 expression was upregulated by AβO-induced oxidative stress and that Prx5 decreased ERK-Drp1-mediated mitochondrial fragmentation and apoptosis of HT-22 neuronal cells. Prx5 expression was affected by AβO, and amelioration of oxidative stress by N-acetyl-L-cysteine decreased AβO-induced Prx5 expression. Prx5 overexpression reduced ROS as well as RNS and apoptotic cell death but Prx5 knockdown did not. In addition, Prx5 overexpression ameliorated ERK-Drp1-mediated mitochondrial fragmentation but Prx5 knockdown did not. These results indicated that inducible Prx5 expression by AβO plays a key role in inhibiting both ERK-Drp1-induced mitochondrial fragmentation and neuronal cell death by regulating oxidative stress. Thus, Prx5 may be a new therapeutic agent for treating AD.

Peroxiredoxin 5 (Prx5) decreases LPS-induced microglial activation through regulation of Ca(2+)/calcineurin-Drp1-dependent mitochondrial fission.

Microglial activation is a hallmark of neurodegenerative diseases. ROS activates microglia by regulating transcription factors to express pro-inflammatory genes and is associated with disruption of Ca2+ homeostasis through thiol redox modulation. Recently, we reported that Prx5 can regulate activation of microglia cells by governing ROS. In addition, LPS leads to excessive mitochondrial fission, and regulation of mitochondrial dynamics involved in a pro-inflammatory response is important for the maintenance of microglial activation. However, the precise relationship among these signals and the role of Prx5 in mitochondrial dynamics and microglial activation is still unknown. In this study, we demonstrated that Ca2+/calcineurin-dependent de-phosphorylation of Drp1 induces mitochondrial fission and regulates mitochondrial ROS production, which influences the expression of pro-inflammatory mediators in LPS-induced microglia cells. Moreover, it is likely that cytosolic and Nox-derived ROS were upstream of mitochondrial fission and mitochondrial ROS generation in activated microglia cells. Prx5 regulates LPS-induced mitochondrial fission through modulation of Ca2+/calcineurin-dependent Drp1 de-phosphorylation by eliminating Nox-derived and cytosolic ROS. Therefore, we suggest that mitochondrial dynamics may be essential for understanding pro-inflammatory responses and that Prx5 may be used as a new therapeutic target to prevent neuroinflammation and neurodegenerative diseases.

Insulin-stimulated lipid accumulation is inhibited by ROS-scavenging chemicals, but not by the Drp1 inhibitor Mdivi-1

Adipocyte differentiation is regulated by intracellular reactive oxygen species (ROS) generation and mitochondrial fission and fusion processes. However, the correlation between intracellular ROS generation and mitochondrial remodeling during adipocyte differentiation is still unknown. Here, we investigated the effect on adipocyte differentiation of 3T3-L1 cells of intracellular ROS inhibition using N-acetyl cysteine (Nac) and Mito-TEMPO and of mitochondrial fission inhibition using Mdivi-1. Differentiated 3T3-L1 adipocytes displayed an increase in mitochondrial fission, ROS generation, and the expression of adipogenic and mitochondrial dynamics-related proteins. ROS scavenger (Nac or Mito-TEMPO) treatment inhibited ROS production, lipid accumulation, the expression of adipogenic and mitochondrial dynamics-related proteins, and mitochondrial fission during adipogenesis of 3T3-L1 cells. On the other hand, treatment with the mitochondrial fission inhibitor Mdivi-1 inhibited mitochondrial fission but did not inhibit ROS production, lipid accumulation, or the expression of adipogenic and mitochondrial dynamics-related proteins, with the exception of phosphorylated Drp1 (Ser616), in differentiated 3T3-L1 adipocytes. The inhibition of mitochondrial fission did not affect adipocyte differentiation, while intracellular ROS production decreased in parallel with inhibition of adipocyte differentiation. Therefore, our results indicated that ROS are an essential regulator of adipocyte differentiation in 3T3-L1 cells.

Neuropeptide Y mitigates ER stress–induced neuronal cell death by activating the PI3K–XBP1 pathway

The unfolded protein response (UPR) is an evolutionarily conserved adaptive reaction that increases cell survival under endoplasmic reticulum (ER) stress conditions. ER stress-associated neuronal cell death pathways play roles in the pathogenesis of neurodegenerative diseases, including Alzheimers, Parkinsons, and Huntingtons disease. Neuropeptide Y (NPY) has an important role in neuroprotection against neurodegenerative diseases. In this study, we investigated whether NPY has a protective role in ER stress-induced neuronal cell death in SK-N-SH human neuroblastoma cells. An ER stress-inducing chemical, tunicamycin, increased the activities of caspase-3 and -4, whereas pretreatment with NPY decreased caspase-3 and -4 activities during the ER stress response. In addition, NPY suppressed the activation of three major ER stress sensors during the tunicamycin-induced ER stress response. NPY-mediated activation of PI3K increased nuclear translocation of XBP1s, which in turn induced expression of Grp78/BiP. Taken together, our data indicated that NPY plays a protective role in ER stress-induced neuronal cell death through activation of the PI3K-XBP1 pathway, and that NPY signaling can serve as therapeutic target for ER stress-mediated neurodegenerative diseases.