Kyounghee Lee

NADPH oxidase 1, a novel molecular source of ROS in hippocampal neuronal death in vascular dementia.

AIMSnChronic cerebral hypoperfusion (CCH) is a common pathological factor that contributes to neurodegenerative diseases such as vascular dementia (VaD). Although oxidative stress has been strongly implicated in the pathogenesis of VaD, the molecular mechanism underlying the selective vulnerability of hippocampal neurons to oxidative damage remains unknown. We assessed whether the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, a specialized superoxide generation system, plays a role in VaD by permanent ligation of bilateral common carotid arteries in rats.nnnRESULTSnMale Wistar rats (10 weeks of age) were subjected to bilateral occlusion of the common carotid arteries (two-vessel occlusion [2VO]). Nox1 expression gradually increased in hippocampal neurons, starting at 1 week after 2VO and for approximately 15 weeks after 2VO. The levels of superoxide, DNA oxidation, and neuronal death in the CA1 subfield of the hippocampus, as well as consequential cognitive impairment, were increased in 2VO rats. Both inhibition of Nox by apocynin, a putative Nox inhibitor, and adeno-associated virus-mediated Nox1 knockdown significantly reduced 2VO-induced reactive oxygen species generation, oxidative DNA damage, hippocampal neuronal degeneration, and cognitive impairment.nnnINNOVATION AND CONCLUSIONnWe provided evidence that neuronal Nox1 is activated in the hippocampus under CCH, causing oxidative stress and consequential hippocampal neuronal death and cognitive impairment. This evidence implies that Nox1-mediated oxidative stress plays an important role in neuronal cell death and cognitive dysfunction in VaD. Nox1 may serve as a potential therapeutic target for VaD.

Role of Neuronal NADPH Oxidase 1 in the Peri-Infarct Regions after Stroke

The molecular mechanism underlying the selective vulnerability of neurons to oxidative damage caused by ischemia—reperfusion (I/R) injury remains unknown. We sought to determine the role of NADPH oxidase 1 (Nox1) in cerebral I/R-induced brain injury and survival of newborn cells in the ischemic injured region. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion. After reperfusion, infarction size, level of superoxide and 8-hydroxy-2′-deoxyguanosine (8-oxo-2dG), and Nox1 immunoreactivity were determined. RNAi-mediated knockdown of Nox1 was used to investigate the role of Nox1 in I/R-induced oxidative damage, neuronal death, motor function recovery, and ischemic neurogenesis. After I/R, Nox1 expression and 8-oxo-2dG immunoreactivity was increased in cortical neurons of the peri-infarct regions. Both infarction size and neuronal death in I/R injury were significantly reduced by adeno-associated virus (AAV)-mediated transduction of Nox1 short hairpin RNA (shRNA). AAV-mediated Nox1 knockdown enhanced functional recovery after MCAO. The level of survival and differentiation of newborn cells in the peri-infarct regions were increased by Nox1 inhibition. Our data suggest that Nox-1 may be responsible for oxidative damage to DNA, subsequent cortical neuronal degeneration, functional recovery, and regulation of ischemic neurogenesis in the peri-infarct regions after stroke.

Effect of 710 nm visible light irradiation on neurite outgrowth in primary rat cortical neurons following ischemic insult

OBJECTIVEnWe previously reported that 710 nm Light-emitting Diode (LED) has a protective effect through cellular immunity activation in the stroke animal model. However, whether LED directly protects neurons suffering from neurodegeneration was entirely unknown. Therefore, we sought to determine the effects of 710 nm visible light irradiation on neuronal protection and neuronal outgrowth in an in vitro stroke model.nnnMATERIALS & METHODSnPrimary cultured rat cortical neurons were exposed to oxygen-glucose deprivation (OGD) and reoxygenation and normal conditions. An LED array with a peak wavelength of 710 nm was placed beneath the covered culture dishes with the room light turned off and were irradiated accordingly. LED treatments (4 min at 4 J/cm(2) and 50 mW/cm(2)) were given once to four times within 8h at 2h intervals for 7 days. Mean neurite density, mean neurite diameter, and total fiber length were also measured after microtubule associated protein 2 (MAP2) immunostaining using the Axio Vision program. Synaptic marker expression and MAPK activation were confirmed by Western blotting.nnnRESULTSnImages captured after MAP2 immunocytochemistry showed significant (p<0.05) enhancement of post-ischemic neurite outgrowth with LED treatment once and twice a day. MAPK activation was enhanced by LED treatment in both OGD-exposed and normal cells. The levels of synaptic markers such as PSD 95, GAP 43, and synaptophysin significantly increased with LED treatment in both OGD-exposed and normal cells (p<0.05).nnnCONCLUSIONnOur data suggest that LED treatment may promote synaptogenesis through MAPK activation and subsequently protect cell death in the in vitro stroke model.

Complete Nonvisualization of Basilar Artery on MR Angiography in Patients with Vertebrobasilar Ischemic Stroke: Favorable Outcome Factors

Background: In vertebrobasilar ischemic stroke, magnetic resonance angiography (MRA) occasionally fails to visualize the basilar artery, but in these patients, little attention has been given to establishing correlations between the clinical and the radiological findings. Our aim was to identify clinical or radiological measures that could assist in predicting a favorable clinical outcome. Methods: Risk factors, clinicoradiological features, and functional outcomes were assessed in 40 patients with vertebrobasilar ischemic stroke whose basilar arteries were absent on MRA. The presence of potential feeding arteries to the posterior circulation was recorded from a review of the MRA data. To permit quantitative analysis of the images, a potential feeding artery score (PFAS; range: 0–8) was established. One point was assigned when a signal was seen from an intracranial vertebral artery, a posterior inferior cerebellar artery, a superior cerebellar artery, or a posterior cerebral artery. On MRI, the location of the infarction was classified as involving the proximal, middle, and distal territories of the intracranial posterior circulation. The infarctions were also categorized as single- or multi-sector infarctions, and according to whether more than one penetrating or branch artery was involved. Clinical outcomes were classified as favorable (modified Rankin Scale = 0–2) or poor (modified Rankin Scale = 3–6). Results: The clinical outcome was favorable in 30% (n = 12) of patients, and poor in 70% (n = 28). A transient ischemic attack preceded the stroke in 48% of patients, especially those with a favorable outcome (67%). Patients with a favorable outcome had a higher PFAS (p = 0.036) and an increased incidence of single-sector infarction (p = 0.049). Conclusions: Our study suggests that a higher PFAS, accompanied by a single-sector infarction, is a predictor of improved clinical outcome in patients with vertebrobasilar ischemic stroke in which the basilar artery was absent on MRA.

DJ-1 Cleavage by Matrix Metalloproteinase 3 Mediates Oxidative Stress-Induced Dopaminergic Cell Death

Oxidative stress is commonly implicated in aging and neurodegenerative conditions such as Parkinsons disease (PD). Mutations in DJ-1 are associated with autosomal recessive early-onset PD. We investigated whether DJ-1 can be degraded in oxidative-stressed dopaminergic neuronal cells, leading to loss of its protective role against oxidative stress. We have shown previously and herein that the active form of matrix metalloproteinase-3 (MMP3) was accumulated in dopamine-producing CATH.a cells in the presence of MPP(+). We show that catalytically active MMP3 cleaved DJ-1, and impaired its antioxidant function. In CATH.a cells, both monomeric and dimeric forms of DJ-1 were diminished in the presence of MPP(+), and this was reversed by MMP3 knockdown or inhibition. While DJ-1 expression was decreased in the substantia nigra of mice administered with MPTP, its degradation was largely attenuated in MMP3 knockout mice. The AKT-signaling pathway, thought to mediate the effect of DJ-1 on cell survival, was also altered. MPP(+) caused decrease in both phospho-Thr308 and phospho-Ser473 forms of AKT, and this was restored by NNGH. Our data suggest that DJ-1 is fragmented by the intracellular MMP3 in response to cell stress, abolishing the protective role of DJ-1 against oxidative damage, and this contributes to the pathogenesis of PD.

Effect of 710-nm Visible Light Irradiation on Neuroprotection and Immune Function after Stroke

Objective: The phototherapeutic effects of low level infrared laser irradiation (808 nm) on brain neuronal cell protection after stroke have been presented recently. We previously reported that 710-nm wavelength visible light (VIS) increases total lymphocyte counts in vivo, especially CD4+ T lymphocytes. In this study, we investigated the effects of 710-nm VIS irradiation on neuronal protection and recovery correlating with cellular immunity in stroke rats. Methods: Rats were subjected to 90-min middle cerebral artery occlusion (MCAO) followed by reperfusion and were divided into two groups: irradiation and no irradiation. The irradiation group had been exposed to 710-nm VIS for 3 weeks after MCAO establishment or sham operation. The helper T cell (CD4+) count in the whole blood and infarct volume were measured. Messenger RNA expression levels of IL-4 and IL-10 in peripheral blood mononuclear cells were measured, a histologic study including microglia activation and regulatory T (Treg) cell markers, neurological severity scoring and a parallel bar walking test were all performed. Results: CD4+ cell count was reduced after MCAO but was significantly increased by 710-nm VIS irradiation. The infarct sizes were decreased in the MCAO + irradiation group compared with the MCAO control group. IL-10 mRNA expression and the immunoreactivity of Treg cells were increased in the MCAO + irradiation group compared with the MCAO control group. Increased microglia activation after MCAO was reduced by 710-nm VIS irradiation. The irradiation group also showed improved neurological severity score levels and step fault scores after MCAO. Conclusions: Our data suggest that 710-nm VIS irradiation may activate cellular immunity, reduce brain infarction and ultimately induce functional recovery in a stroke animal model.

Arabidopsis ATXR2 deposits H3K36me3 at the promoters of LBD genes to facilitate cellular dedifferentiation

The histone lysine methyltransferase ATXR2 promotes cellular dedifferentiation in Arabidopsis thaliana. Epigenetic control of dedifferentiation Some plant cells can dedifferentiate to form a mass of pluripotent cells called callus. This not only occurs at wound sites but can also be induced by specific laboratory culture conditions. Lee et al. found that the histone lysine methyltransferase ATXR2 promoted cellular dedifferentiation during callus formation in Arabidopsis thaliana by stimulating the expression of LBD genes, which encode transcription factors that promote cell cycle progression. ATXR2 localized to LBD promoters, stimulated the accumulation of lysine-methylated histones at these promoters, and was recruited to the promoters by the transcription factors ARF7 and ARF19. Epigenetic regulation is a key mechanism controlling cell potency and differentiation in both plants and animals, and these findings contribute to understanding the remarkable developmental plasticity of plant cells. Cellular dedifferentiation, the transition of differentiated somatic cells to pluripotent stem cells, ensures developmental plasticity and contributes to wound healing in plants. Wounding induces cells to form a mass of unorganized pluripotent cells called callus at the wound site. Explanted cells can also form callus tissues in vitro. Reversible cellular differentiation-dedifferentiation processes in higher eukaryotes are controlled mainly by chromatin modifications. We demonstrate that ARABIDOPSIS TRITHORAX-RELATED 2 (ATXR2), a histone lysine methyltransferase that promotes the accumulation of histone H3 proteins that are trimethylated on lysine 36 (H3K36me3) during callus formation, promotes early stages of cellular dedifferentiation through activation of LATERAL ORGAN BOUNDARIES DOMAIN (LBD) genes. The LBD genes of Arabidopsis thaliana are activated during cellular dedifferentiation to enhance the formation of callus. Leaf explants from Arabidopsis atxr2 mutants exhibited a reduced ability to form callus and a substantial reduction in LBD gene expression. ATXR2 bound to the promoters of LBD genes and was required for the deposition of H3K36me3 at these promoters. ATXR2 was recruited to LBD promoters by the transcription factors AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19. Leaf explants from arf7-1arf19-2 double mutants were defective in callus formation and showed reduced H3K36me3 accumulation at LBD promoters. Genetic analysis provided further support that ARF7 and ARF19 were required for the ability of ATXR2 to promote the expression of LBD genes. These observations indicate that the ATXR2-ARF-LBD axis is key for the epigenetic regulation of callus formation in Arabidopsis.

Dynamic Epigenetic Changes during Plant Regeneration

Plants have the remarkable ability to drive cellular dedifferentiation and regeneration. Changes in epigenetic landscapes accompany the cell fate transition. Notably, modifications of chromatin structure occur primarily during callus formation via an in vitro tissue culture process and, thus, pluripotent callus cells have unique epigenetic signatures. Here, we highlight the latest progress in epigenetic regulation of callus formation in plants, which addresses fundamental questions related to cell fate changes and pluripotency establishment. Global and local modifications of chromatin structure underlie callus formation, and the combination and sequence of epigenetic modifications further shape intricate cell fate changes. This review illustrates how a series of chromatin marks change dynamically during callus formation and their biological relevance in plant regeneration.

The Circadian Clock Sets the Time of DNA Replication Licensing to Regulate Growth in Arabidopsis

The circadian clock and cell cycle as separate pathways have been well documented in plants. Elucidating whether these two oscillators are connected is critical for understanding plant growth. We found that a slow-running circadian clock decelerates the cell cycle and, conversely, a fast clock speeds it up. The clock component TOC1 safeguards the G1-to-S transition and controls the timing of the mitotic cycle at early stages of leaf development. TOC1 also regulates somatic ploidy at later stages of leaf development and in hypocotyl cells. The S-phase is shorter and delayed in TOC1 overexpressing plants, which correlates with the diurnal repression of the DNA replication licensing gene CDC6 through binding of TOC1 to the CDC6 promoter. The slow cell-cycle pace in TOC1-ox also results in delayed tumor progression in inflorescence stalks. Thus, TOC1 sets the time of the DNA pre-replicative machinery to control plant growth in resonance with the environment.

The HAF2 protein shapes histone acetylation levels of PRR5 and LUX loci in Arabidopsis

Main conclusionThe histone acetyltransferase HAF2 facilitates H3 acetylation deposition at the PRR5 and LUX promoters to contribute to robust circadian oscillation.The circadian clock ensures synchronization of endogenous rhythmic processes with environmental cycles. Multi-layered regulation underlies precise circadian oscillation, and epigenetic regulation is emerging as a crucial scheme for robust circadian maintenance. Here, we report that HISTONE ACETYLTRANSFERASE OF THE TAFII250 FAMILY 2 (HAF2) is involved in circadian homeostasis. The HAF2 gene is activated at midday, and its temporal expression is shaped by CIRCADIAN CLOCK-ASSOCIATED 1. The midday-activated HAF2 protein stimulates H3 acetylation (H3ac) deposition at the PRR5 and LUX loci, contributing to establishment of the raising phase. These results indicate that epigenetic waves in circadian networks underlie temporal compartmentalization of circadian components and stable maintenance of circadian oscillation.