Byung Kwan Jin

Thrombin-Induced Oxidative Stress Contributes to the Death of Hippocampal Neurons In Vivo: Role of Microglial NADPH Oxidase

The present study investigated whether thrombin, a potent microglial activator, can induce reactive oxygen species (ROS) generation through activation of microglial NADPH oxidase and if this may contribute to oxidative damage and consequent neurodegeneration. Seven days after intrahippocampal injection of thrombin, Nissl staining and immunohistochemistry using the neuronal-specific nuclear protein NeuN revealed a significant loss in hippocampal CA1 neurons. In parallel, thrombin-activated microglia, assessed by OX-42 and OX-6 immunohistochemistry, and ROS production, assessed by hydroethidine histochemistry, were observed in the hippocampal CA1 area in which degeneration of hippocampal neurons occurred. Reverse transcription-PCR at various time points after thrombin administration demonstrated an early and transient expression of inducible nitric oxide synthase (iNOS) and several proinflammatory cytokines. Western blot analysis and double-label immunohistochemistry showed an increase in the expression of and the localization of iNOS within microglia. Additional studies demonstrated that thrombin induced the upregulation of membrane (gp91phox) and cytosolic (p47phox and p67phox) components, translocation of cytosolic proteins (p47phox, p67phox, and Rac1) to the membrane, and p67phox expression of the NADPH oxidase in microglia in the hippocampus in vivo, indicating the activation of NADPH oxidase. The thrombin-induced oxidation of proteins and loss of hippocampal CA1 neurons were partially inhibited by an NADPH oxidase inhibitor and by an antioxidant. To our knowledge, the present study is the first to demonstrate that thrombin-induced neurotoxicity in the hippocampus in vivo is caused by microglial NADPH oxidase-mediated oxidative stress. This suggests that thrombin inhibition or enhancing antioxidants may be beneficial for the treatment of neurodegenerative diseases, such as Alzheimers disease, that are associated with microglial-derived oxidative damage.

Transient Receptor Potential Vanilloid Subtype 1 Mediates Cell Death of Mesencephalic Dopaminergic Neurons In Vivo and In Vitro

Intranigral injection of the transient receptor potential vanilloid subtype 1 (TRPV1; also known as VR1) agonist capsaicin (CAP) into the rat brain, or treatment of rat mesencephalic cultures with CAP, resulted in cell death of dopaminergic (DA) neurons, as visualized by immunocytochemistry. This in vivo and in vitro effect was ameliorated by the TRPV1 antagonist capsazepine (CZP) or iodo-resiniferatoxin, suggesting the direct involvement of TRPV1 in neurotoxicity. In cultures, both CAP and anandamide (AEA), an endogenous ligand for both TRPV1 and cannabinoid type 1 (CB1) receptors, induced degeneration of DA neurons, increases in intracellular Ca2+ ([Ca2+]i), and mitochondrial damage, which were inhibited by CZP, the CB1 antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) or the intracellular Ca2+ chelator BAPTA/AM. We also found that CAP or AEA increased mitochondrial cytochrome c release as well as immunoreactivity to cleaved caspase-3 and that the caspase-3 inhibitor z-Asp-Glu-Val-Asp-fmk protected DA neurons from CAP- or AEA-induced neurotoxicity. Additional studies demonstrated that treatment of mesencephalic cultures with CB1 receptor agonist (6aR)-trans 3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d] pyran-9-methanol (HU210) also produced degeneration of DA neurons and increases in [Ca2+]i, which were inhibited by AM251 and BAPTA/AM. The CAP-, AEA-, or HU210-induced increases in [Ca2+]i were dependent on extracellular Ca2+, with significantly different patterns of Ca2+ influx. Surprisingly, CZP and AM251 reversed HU210- or CAP-induced neurotoxicity by inhibiting Ca2+ influx, respectively, suggesting the existence of functional cross talk between TRPV1 and CB1 receptors. To our knowledge, this study is the first to demonstrate that the activation of TRPV1 and/or CB1 receptors mediates cell death of DA neurons. Our findings suggest that these two types of receptors, TRPV1 and CB1, may contribute to neurodegeneration in response to endogenous ligands such as AEA.

Cleavage of Bax is mediated by caspase‐dependent or ‐independent calpain activation in dopaminergic neuronal cells: protective role of Bcl‐2

Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress‐specific protease activation pathway exists, and to what extent Bcl‐2 plays a role in preventing drug‐induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase‐dependent apoptosis while a dopaminergic neurotoxin, MPP+ largely induced caspase‐independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS‐ and MPP+‐induced cell death, Bax was cleaved into an 18‐kDa fragment. This 18‐kDa fragment appeared only in the mitochondria‐enriched heavy membrane fraction of STS‐treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP+‐treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [35S]methionine‐labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP+‐ and STS‐induced Bax cleavage. Intriguingly, overexpression of baculovirus‐derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS‐ but not MPP+‐induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP+‐induced but not from STS‐induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl‐2 prevented both STS‐ and MPP+‐induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl‐2 may play a protective role by primarily blocking drug‐induced caspase or calpain activity in dopaminergic neuronal cells.

Paroxetine Prevents Loss of Nigrostriatal Dopaminergic Neurons by Inhibiting Brain Inflammation and Oxidative Stress in an Experimental Model of Parkinson’s Disease

The present study examined whether the antidepressant paroxetine promotes the survival of nigrostriatal dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease. MPTP induced degeneration of nigrostriatal DA neurons and glial activation as visualized by tyrosine hydroxylase, macrophage Ag complex-1, and/or glial fibrillary acidic protein immunoreactivity. Real-time PCR, Western blotting, and immunohistochemistry showed upregulation of proinflammatory cytokines, activation of microglial NADPH oxidase and astroglial myeloperoxidase, and subsequent reactive oxygen species production and oxidative DNA damage in the MPTP-treated substantia nigra. Treatment with paroxetine prevented degeneration of nigrostriatal DA neurons, increased striatal dopamine levels, and improved motor function. This neuroprotection afforded by paroxetine was associated with the suppression of astroglial myeloperoxidase expression and/or NADPH oxidase-derived reactive oxygen species production and reduced expression of proinflammatory cytokines, including IL-1β, TNF-α, and inducible NO synthase, by activated microglia. The present findings show that paroxetine may possess anti-inflammatory properties and inhibit glial activation-mediated oxidative stress, suggesting that paroxetine and its analogues may have therapeutic value in the treatment of aspects of Parkinson’s disease related to neuroinflammation.

Inhibition of thrombin-induced microglial activation and NADPH oxidase by minocycline protects dopaminergic neurons in the substantia nigra in vivo

The present study shows that activation of microglial NADPH oxidase and production of reactive oxygen species (ROS) is associated with thrombin‐induced degeneration of nigral dopaminergic neurons in vivo. Seven days after thrombin injection in the rat substantia nigra (SN), tyrosine hydroxylase immunocytochemistry showed a significant loss of nigral dopaminergic neurons. This cell death was accompanied by localization of terminal deoxynucleotidyl transferase‐mediated fluorecein UTP nick‐end labelling (TUNEL) staining within dopaminergic neurons. This neurotoxicity was antagonized by the semisynthetic tetracycline derivative, minocycline, and the observed neuroprotective effects were associated with the ability of minocycline to suppress NADPH oxidase‐derived ROS production and pro‐inflammatory cytokine expression, including interleukin‐1β and inducible nitric oxide synthase, from activated microglia. These results suggest that microglial NADPH oxidase may be a viable target for neuroprotection against oxidative damage.

Neuroprotective effect of nicotine on dopaminergic neurons by anti-inflammatory action.

Epidemiological studies have reported that smoking is associated with a lower incidence of Parkinsons disease (PD), leading to theories that smoking in general and nicotine in particular might be neuroprotective. Recent studies suggested cholinergic anti‐inflammatory pathway‐regulating microglial activation through α7 nicotinic receptors. In the present study, we used lipopolysaccharide (LPS)‐induced in vitro and in vivo inflammation models to investigate whether nicotine has a protective effect on the dopaminergic system through an anti‐inflammatory mechanism. Nicotine pretreatment considerably decreased microglial activation with significant reduction of tumour necrosis factor (TNF)‐α mRNA expression and TNF‐α release induced by LPS stimulation. In co‐cultures of microglia and mesencephalic neurons, nicotine pretreatment significantly decreased the loss of tyrosine hydroxylase‐immunopositive (TH‐ip) cells, approximately twice more than the LPS‐only treatment. α‐Bungarotoxin, an α7 nicotinic acetylcholine receptor subunit‐selective blocker, considerably blocked the inhibitory effects of nicotine on microglial activation and TH‐ip neuronal loss. Chronic nicotine pretreatment in rats showed that TH‐ip neuronal loss induced by LPS stimulation in the substantia nigra was dramatically decreased, which was clearly accompanied by a reduction in the formation of TNF‐α. The present study demonstrated that nicotine has a neuroprotective effect on dopaminergic neurons via an anti‐inflammatory mechanism mediated by the modulation of microglial activation. Along with various neuroprotective effects of nicotine, the anti‐inflammatory mechanism of nicotine could have a major therapeutic implication in the preventive treatment of PD.

Fluoxetine prevents MPTP-induced loss of dopaminergic neurons by inhibiting microglial activation

Parkinsons disease (PD) is characterized by degeneration of nigrostriatal dopaminergic (DA) neurons. Mice treated with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) exhibit microglial activation-induced oxidative stress and inflammation, and nigrostriatal DA neuronal damage, and thus serve as an experimental model of PD. Here, we report that fluoxetine, one of the most commonly prescribed antidepressants, prevents MPTP-induced degeneration of nigrostriatal DA neurons and increases striatal dopamine levels with the partial motor recovery. This was accompanied by inhibiting transient expression of proinflammatory cytokines and inducible nitric oxide synthase; and attenuating microglial NADPH oxidase activation, reactive oxygen species/reactive nitrogen species production, and consequent oxidative damage. Interestingly, fluoxetine was found to protect DA neuronal damage from 1-methyl-4-phenyl-pyridinium (MPP(+)) neurotoxicity in co-cultures of mesencephalic neurons and microglia but not in neuron-enriched mesencephalic cultures devoid of microglia. The present in vivo and in vitro findings show that fluoxetine may possess anti-inflammatory properties and inhibit glial activation-mediated oxidative stress. Therefore, we carefully propose that neuroprotection of fluoxetine might be associated with its anti-inflammatory properties and could be employed as novel therapeutic agents for PD and other disorders associated with neuroinflammation and microglia-derived oxidative damage.

Oxidative modification of peroxiredoxin is associated with drug-induced apoptotic signaling in experimental models of Parkinson disease

The aim of this study was to investigate changes in protein profiles during the early phase of dopaminergic neuronal death using two-dimensional gel electrophoresis in conjunction with mass spectrometry. Several protein spots were identified whose expression was significantly altered following treatment of MN9D dopaminergic neuronal cells with 6-hydroxydopamine (6-OHDA). In particular, we detected oxidative modification of thioredoxin-dependent peroxidases (peroxiredoxins; PRX) in treated MN9D cells. Oxidative modification of PRX induced by 6-OHDA was blocked in the presence of N-acetylcysteine, suggesting that reactive oxygen species (ROS) generated by 6-OHDA induce oxidation of PRX. These findings were confirmed in primary cultures of mesencephalic neurons and in rat brain injected stereotaxically. Overexpression of PRX1 in MN9D cells (MN9D/PRX1) exerted neuroprotective effects against death induced by 6-OHDA through scavenging of ROS. Consequently, generation of both superoxide anion and hydrogen peroxide following 6-OHDA treatment was decreased in MN9D/PRX1. Furthermore, overexpression of PRX1 protected cells against 6-OHDA-induced activation of p38 MAPK and subsequent activation of caspase-3. In contrast, 6-OHDA-induced apoptotic death signals were enhanced by RNA interference-targeted reduction of PRX1 in MN9D cells. Taken together, our data suggest that the redox state of PRX may be intimately involved in 6-OHDA-induced dopaminergic neuronal cell death and also provide a molecular mechanism by which PRX1 exerts a protective role in experimental models of Parkinson disease.

Microglia Expressing Interleukin-13 Undergo Cell Death and Contribute to Neuronal Survival In Vivo

How to minimize brain inflammation is pathophysiologically important, since inflammation induced by microglial activation can exacerbate brain damage. In the present report, we show that injection of lipopolysaccharide (LPS) into the rat cortex led to increased levels of interleukin‐13 (IL‐13) and to IL‐13 immunoreactivity, followed by the substantial loss of microglia at 3 days post‐LPS. IL‐13 levels in LPS‐injected cortex reached a peak at 12 h post‐injection, remained elevated at 24 h, and returned to basal levels at day 4. In parallel, IL‐13 immunoreactivity was detected as early as 12 h post‐LPS and maintained up to 24 h; it disappeared at 4 days. Surprisingly, IL‐13 immunoreactivity was detected exclusively in microglia, but not in neurons or astrocytes. Following treatment with LPS in vitro, IL‐13 expression was also induced in microglia in the presence of neurons, but not in the presence of astrocytes or in cultured pure microglia alone. In experiments designed to determine the involvement of IL‐13 in microglia cell death, IL‐13‐neutralizing antibodies significantly increased survival of activated microglia at 3 days post‐LPS. Consistent with these results, the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor‐α (TNF‐α) was sustained in activated microglia and neuronal cell death was consequently increased. Taken together, the present study is the first to demonstrate the endogenous expression of IL‐13 in LPS‐activated microglia in vivo, and to demonstrate that neurons may be required for IL‐13 expression in microglia. Our data strongly suggest that IL‐13 may control brain inflammation by inducing the death of activated microglia in vivo, resulting in an enhancement of neuronal survival.

Melatonin protects nigral dopaminergic neurons from 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity in rats

In the present study, the in vivo neuroprotective effects of melatonin, as an antioxidant, were assessed in Sprague-Dawley rats with a unilateral lesion of substantia nigra (SN) caused by a stereotaxic injection of neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). When expressed as a percentage ratio of lesioned to intact side, increased lipid peroxidation product (malondialdehyde, MDA, 117% of control) and decreased tyrosine hydroxylase (TH) enzyme activity (60% of control) in SN were observed 4 h after MPP+ lesion. In contrast, however, melatonin treatment prevented MPP+ neurotoxicity by the almost complete recovery of MDA (99% of control) and TH levels (96% of control), indicating the potent antioxidative effects of melatonin. In addition, further reduction of TH enzyme activity (52% of control) was seen 1 week after MPP+ infusion. Continuous (twice a day for 5 days), not acute (4 h) treatment with melatonin produced the partial, but not statistically significant, recovery of TH enzyme activity (71% of control), when sacrificed 1 week after MPP+ lesion. Taken together, the present results support the hypothesis that melatonin may provide the useful therapeutic strategies for the treatment of oxidative stress-induced neurodegenerative disease such as Parkinsons disease (PD).