Dong-Hee Choi

Transglutaminase 2 induces NF-κB activation via a novel pathway in BV-2 microglia

Transglutaminase 2 (TGase 2) expression is increased in inflammatory diseases. We demonstrated previously that inhibitors of TGase 2 reduce nitric oxide (NO) generation in a lipopolysaccharide (LPS)-treated microglial cell line. However, the precise mechanism by which TGase 2 promotes inflammation remains unclear. We found that TGase 2 activates the transcriptional activator nuclear factor (NF)-κB and thereby enhances LPS-induced expression of inducible nitric-oxide synthase. TGase 2 activates NF-κB via a novel pathway. Rather than stimulating phosphorylation and degradation of the inhibitory subunit α of NF-κB (I-κBα), TGase2 induces its polymerization. This polymerization results in dissociation of NF-κB and its translocation to the nucleus, where it is capable of up-regulating a host of inflammatory genes, including inducible nitric-oxide synthase and tumor necrosis factor α (TNF-α). Indeed, TGase inhibitors prevent depletion of monomeric I-κBα in the cytosol of cells overexpressing TGase 2. In an LPS-induced rat brain injury model, TGase inhibitors significantly reduced TNF-α synthesis. The findings are consistent with a model in which LPS-induced NF-κB activation is the result of phosphorylation of I-κBα by I-κB kinase as well as I-κBα polymerization by TGase 2. Safe and stable TGase2 inhibitors may be effective agents in diseases associated with inflammation.

NADPH Oxidase 1-Mediated Oxidative Stress Leads to Dopamine Neuron Death in Parkinson's Disease

AIM Oxidative stress has long been considered as a major contributing factor in the pathogenesis of Parkinsons disease. However, molecular sources for reactive oxygen species in Parkinsons disease have not been clearly elucidated. Herein, we sought to investigate whether a superoxide-producing NADPH oxidases (NOXs) are implicated in oxidative stress-mediated dopaminergic neuronal degeneration. RESULTS Expression of various Nox isoforms and cytoplasmic components were investigated in N27, rat dopaminergic cells. While most of Nox isoforms were constitutively expressed, Nox1 expression was significantly increased after treatment with 6-hydroxydopamine. Rac1, a key regulator in the Nox1 system, was also activated. Striatal injection of 6-hydroxydopamine increased Nox1 expression in dopaminergic neurons in the rat substantia nigra. Interestingly, it was localized into the nucleus, and immunostaining for DNA oxidative stress marker, 8-oxo-dG, was increased. Nox1 expression was also found in the nucleus of dopaminergic neurons in the substantia nigra of Parkinsons disease patients. Adeno-associated virus-mediated Nox1 knockdown or Rac1 inhibition reduced 6-hydroxydopamine-induced oxidative DNA damage and dopaminergic neuronal degeneration significantly. INNOVATION Nox1/Rac1 could serve as a potential therapeutic target for Parkinsons disease. CONCLUSION We provide evidence that dopaminergic neurons are equipped with the Nox1/Rac1 superoxide-generating system. Stress-induced Nox1/Rac1 activation causes oxidative DNA damage and neurodegeneration. Reduced dopaminergic neuronal death achieved by targeting Nox1/Rac1, emphasizes the impact of oxidative stress caused by this system on the pathogenesis and therapy in Parkinsons disease.

Synergistic Memory Impairment Through the Interaction of Chronic Cerebral Hypoperfusion and Amlyloid Toxicity in a Rat Model

Background and Purpose— Vascular pathology and Alzheimer disease (AD) pathology have been shown to coexist in the brains of dementia patients. We investigated how cognitive impairment could be exacerbated in a rat model of combined injury through the interaction of chronic cerebral hypoperfusion and amyloid beta (A&bgr;) toxicity. Methods— In Wistar rats, chronic cerebral hypoperfusion was modeled by permanent occlusion of bilateral common carotid arteries (BCCAo). Further, AD pathology was modeled by bilateral intracerebroventricular A&bgr; (A&bgr; toxicity) using a nonphysiological A&bgr; peptide (A&bgr; 25 to 35). The experimental animals were divided into 4 groups, including sham, single injury (A&bgr; toxicity or BCCAo), and combined injury (BCCAo-A&bgr; toxicity) groups (n=7 per group) . Cerebral blood flow and metabolism were measured using small animal positron emission tomography. A Morris water maze task, novel object location and recognition tests, and histological investigation, including neuronal cell death, apoptosis, neuroinflammation, and AD-related pathology, were performed. Results— Spatial memory impairment was synergistically exacerbated in the BCCAo–A&bgr; toxicity group as compared to the BCCAo or A&bgr; toxicity groups (P<0.05). Compared to the sham group, neuroinflammation with microglial or astroglial activation was increased both in multiple white matter lesions and the hippocampus in other experimental groups. AD-related pathology was enhanced in the BCCAo–A&bgr; toxicity group compared to the A&bgr; toxicity group. Conclusion— Our experimental results support a clinical hypothesis of the deleterious interaction between chronic cerebral hypoperfusion and A&bgr; toxicity. Chronic cerebral hypoperfusion-induced perturbation in the equilibrium of AD-related pathology may exacerbate cognitive impairment in a rat model of combined injury.

Role of Matrix Metalloproteinase 3-mediated α-Synuclein Cleavage in Dopaminergic Cell Death

Evidence suggests that the C-terminal truncation of α-synuclein is equally important as aggregation of α-synuclein in Parkinson disease (PD). Our previous results showed that an endopeptidase, matrix metalloproteinase-3 (MMP3), was induced and activated in dopaminergic (DA) cells upon stress conditions. Here, we report that MMP3 cleaved α-synuclein in vitro and in vivo and that α-synuclein and MMP3 were co-localized in Lewy bodies (LB) in the postmortem brains of PD patients. Incubation of α-synuclein with the catalytic domain of MMP3 (cMMP3) resulted in generation of several peptides, and the peptide profiles of WT α-synuclein (WTsyn) and A53T mutant (A53Tsyn) were different. Combined analysis using mass spectrometry and N-terminal determination revealed that MMP3 generated C-terminally truncated peptides of amino acids 1–78, 1–91, and 1–93 and that A53Tsyn produced significantly higher quantities of these peptides. Similar sizes of peptides were detected in N27 DA cells under oxidative stress and RNA interference to knock down MMP3-attenuated peptide generation. Co-overexpression of cMMP3 with either WTsyn or A53Tsyn led to a reduction in Triton X-100-insoluble aggregates and an increase in protofibril-like small aggregates. In addition, overexpression of the 1–93-amino acid peptide in the substantia nigra led to DA neuronal loss without LB-like aggregate formation. The results strongly indicate that MMP3 digestion of α-synuclein in DA neurons plays a pivotal role in the progression of PD through modulation of α-synuclein in aggregation, LB formation, and neurotoxicity.

NADPH Oxidase 1 Mediates α-Synucleinopathy in Parkinson's Disease

Accumulation of misfolded α-synuclein is the pathological hallmark of Parkinsons disease (PD). Nevertheless, little is known about the mechanism contributing to α-synuclein aggregation and its further toxicity to dopaminergic neurons. Since oxidative stress can increase the expression and aggregation levels of α-synuclein, NADPH oxidases (Noxs), which are responsible for reactive oxygen species generation, could be major players in α-synucleinopathy. Previously, we demonstrated that Nox1 is expressed in dopaminergic neurons of the PD animal models as well as postmortem brain tissue of PD patients, and is responsible for oxidative stress and subsequent neuronal degeneration. Here, using paraquat (PQ)-based in vitro and in vivo PD models, we show that Nox1 has a crucial role in modulating the behavior of α-synuclein expression and aggregation in dopaminergic neurons. We observed in differentiated human dopaminergic cells that Nox1 and α-synuclein expressions are increased under PQ exposure. Nox1 knockdown significantly reduced both α-synuclein expression and aggregation, supporting the role of Nox1 in this process. Furthermore, in rats exposed to PQ, the selective knockdown of Nox1 in the substantia nigra, using adeno-associated virus encoding Nox1-specific shRNA, largely attenuated the PQ-mediated increase of α-synuclein and ubiquitin expression levels as well as α-synuclein aggregates (proteinase K resistant) and A11 oligomers. Significant reductions in oxidative stress level and dopaminergic neuronal loss were also observed. Our data reveal a new mechanism by which α-synuclein becomes a neuropathologic protein through Nox1-mediated oxidative stress. This finding may be used to generate new therapeutic interventions that slower the rate of α-synuclein aggregation and the progression of PD pathogenesis.

Subchronic Treatment of Donepezil Rescues Impaired Social, Hyperactive, and Stereotypic Behavior in Valproic Acid-Induced Animal Model of Autism

Autism spectrum disorder (ASD) is a group of pervasive developmental disorders with core symptoms such as sociability deficit, language impairment, and repetitive/restricted behaviors. Although worldwide prevalence of ASD has been increased continuously, therapeutic agents to ameliorate the core symptoms especially social deficits, are very limited. In this study, we investigated therapeutic potential of donepezil for ASD using valproic acid-induced autistic animal model (VPA animal model). We found that prenatal exposure of valproic acid (VPA) induced dysregulation of cholinergic neuronal development, most notably the up-regulation of acetylcholinesterase (AChE) in the prefrontal cortex of affected rat and mouse offspring. Similarly, differentiating cortical neural progenitor cell in culture treated with VPA showed increased expression of AChE in vitro. Chromatin precipitation experiments revealed that acetylation of histone H3 bound to AChE promoter region was increased by VPA. In addition, other histone deacetyalse inhibitors (HDACIs) such as trichostatin A and sodium butyrate also increased the expression of AChE in differentiating neural progenitor cells suggesting the essential role of HDACIs in the regulation of AChE expression. For behavioral analysis, we injected PBS or donepezil (0.3 mg/kg) intraperitoneally to control and VPA mice once daily from postnatal day 14 all throughout the experiment. Subchronic treatment of donepezil improved sociability and prevented repetitive behavior and hyperactivity of VPA-treated mice offspring. Taken together, these results provide evidence that dysregulation of ACh system represented by the up-regulation of AChE may serve as an effective pharmacological therapeutic target against autistic behaviors in VPA animal model of ASD, which should be subjected for further investigation to verify the clinical relevance.

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

AIMS Chronic 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. RESULTS Male 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. INNOVATION AND CONCLUSION We 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.

Characterization of White Matter Injury in a Rat Model of Chronic Cerebral Hypoperfusion

Background and Purpose— Chronic cerebral hypoperfusion can lead to ischemic white matter injury resulting in vascular dementia. To characterize white matter injury in vascular dementia, we investigated disintegration of diverse white matter components using a rat model of chronic cerebral hypoperfusion. Methods— Chronic cerebral hypoperfusion was modeled in Wistar rats by permanent occlusion of the bilateral common carotid arteries. We performed cognitive behavioral tests, including the water maze task, odor discrimination task, and novel object test; histological investigation of neuroinflammation, oligodendrocytes, myelin basic protein, and nodal or paranodal proteins at the nodes of Ranvier; and serial diffusion tensor imaging. Cilostazol was administered to protect against white matter injury. Results— Diverse cognitive impairments were induced by chronic cerebral hypoperfusion. Disintegration of white matter was characterized by neuroinflammation, loss of oligodendrocytes, attenuation of myelin density, structural derangement at the nodes of Ranvier, and disintegration of white matter tracts. Cilostazol protected against cognitive impairments and white matter disintegration. Conclusions— White matter injury induced by chronic cerebral hypoperfusion can be characterized by disintegration of diverse white matter components. Cilostazol might be a therapeutic strategy against white matter disintegration in patients with vascular dementia.

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

OBJECTIVE We 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. MATERIALS & METHODS Primary 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. RESULTS Images 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). CONCLUSION Our data suggest that LED treatment may promote synaptogenesis through MAPK activation and subsequently protect cell death in the in vitro stroke model.