Onyou Hwang

Role of Oxidative Stress in Parkinson's Disease

Parkinsons disease (PD) is a progressive neurodegenerative movement disorder associated with a selective loss of the dopamine(DA)rgic neurons in the substantia nigra pars compacta and the degeneration of projecting nerve fibers in the striatum. Because there is currently no therapy that delays the neurodegenerative process, modification of the disease course by neuroprotective therapy is an important unmet clinical need. Toward this end, understanding cellular mechanisms that render the nigral neurons particularly vulnerable have been a subject of intensive research. Increasing evidence suggests that oxidative stress plays a major role. The metabolism of DA itself contributes to oxidative stress, resulting in modification of intracellular macromolecules whose functions are important for cell survival. Mitochondrial dysfunction and the consequent increase in reactive oxygen species also trigger a sequence of events that leads to cell demise. In addition, activated microglia produce nitric oxide and superoxide during neuroinflammatory responses, and this is aggravated by the molecules released by damaged DAergic neurons such as α-synuclein, neuromelanin and matrix metalloproteinase-3. Ways to reduce oxidative stress therefore can provide a therapeutic strategy. NAD(P)H:quinone reductase (NQO1) and other antioxidant enzymes, whose gene expression are commonly under the regulation of the transcription factor Nrf2, can serve as target proteins utilized toward development of disease-modifying therapy for PD.

Matrix Metalloproteinase-3: A Novel Signaling Proteinase from Apoptotic Neuronal Cells That Activates Microglia

Microglial activation and inflammation are associated with progressive neuronal apoptosis in neurodegenerative human brain disorders. We sought to investigate molecular signaling mechanisms that govern activation of microglia in apoptotic neuronal degeneration. We report here that the active form of matrix metalloproteinase-3 (MMP-3) was released into the serum-deprived media (SDM) of PC12 cells and other media of apoptotic neuronal cells within 2-6 h of treatment of the cells, and SDM and catalytic domain of recombinant MMP-3 (cMMP-3) activated microglia in primary microglia cultures as well as BV2 cells, a mouse microglia cell line. Both SDM and cMMP-3 induced generation of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), IL-1β, and interleukin-1 receptor antagonist but not IL-12 and inducible nitric oxide synthase, which are readily induced by lipopolysaccharide, in microglia, suggesting that there is a characteristic pattern of microglial cytokine induction by apoptotic neurons. Neither glial cell line-derived neurotrophic factor nor anti-inflammatory cytokines, such as IL-10 and transforming growth factor-β1, were induced. SDM and cMMP-3 extensively released TNF-α from microglia and activated the nuclear factor-κB pathway, and these microglial responses were totally abolished by preincubation with an MMP-3 inhibitor, NNGH [N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid]. MMP-3-mediated microglial activation mostly depended on ERK (extracellular signal-regulated kinase) phosphorylation but not much on either JNK (c-Jun N-terminal protein kinase) or p38 activation. Conditioned medium of SDM- or cMMP-3-activated BV2 cells caused apoptosis of PC12 cells. These results strongly suggest that the distinctive signal of neuronal apoptosis is the release of active form of MMP-3 that activates microglia and subsequently exacerbates neuronal degeneration. Therefore, the release of MMP-3 from apoptotic neurons may play a major role in degenerative human brain disorders, such as Parkinsons disease.

A pivotal role of matrix metalloproteinase-3 activity in dopaminergic neuronal degeneration via microglial activation

Recent studies have demonstrated that activated microglia play an important role in dopamine (DA) neuronal degeneration in Parkinson disease (PD) by generating NADPH‐oxidase (NADPHO)‐derived superoxide. However, the molecular mechanisms that underlie microglial activation in DA cell death are still disputed. We report here that matrix metalloproteinase‐3 (MMP‐3) was newly induced and activated in stressed DA cells, and the active form of MMP‐3 (actMMP‐3) was released into the medium. The released actMMP‐3, as well as catalytically active recombinant MMP‐3 (cMMP‐3) led to microglial activation and superoxide generation in microglia and enhanced DA cell death. cMMP‐3 caused DA cell death in mesencephalic neuron‐glia mixed culture of wild‐type (WT) mice, but this was attenuated in the culture of NADPHO subunit null mice (gp91phox‐/‐), suggesting that NADPHO mediated the cMMP‐3‐induced microglial production of superoxide and DA cell death. Furthermore, in the N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐injected animal model of PD, nigrostriatal DA neuronal degeneration, microglial activation, and superoxide generation were largely attenuated in MMP‐3‐/‐mice. These results indicate that actMMP‐3 released from stressed DA neurons is responsible for microglial activation and generation of NADPHO‐derived superoxide and eventually enhances nigrostriatal DA neuronal degeneration. Our results could lead to a novel therapeutic approach to PD. Kim, Y. S., Choi, D. H., Block, M. L., Lorenzl, S., Yang, L., Kim, Y. J., Sugama, S., Cho, B. P., Ywang, O., Browne, S. E., Kim, S. Y., Hong, J.‐S., Beal, M. F., Jon, T. H. A pivotal role of matrix metalloproteinase‐3 activity in dopaminergic neuronal degeneration via microglial activation. FASEB J. 21, 179–187 (2007)

Impact of Circadian Nuclear Receptor REV-ERBα on Midbrain Dopamine Production and Mood Regulation

The circadian nature of mood and its dysfunction in affective disorders is well recognized, but the underlying molecular mechanisms are still unclear. Here, we show that the circadian nuclear receptor REV-ERBα, which is associated with bipolar disorder, impacts midbrain dopamine production and mood-related behavior in mice. Genetic deletion of the Rev-erbα gene or pharmacological inhibition of REV-ERBα activity in the ventral midbrain induced mania-like behavior in association with a central hyperdopaminergic state. Also, REV-ERBα repressed tyrosine hydroxylase (TH) gene transcription via competition with nuclear receptor-related 1 protein (NURR1), another nuclear receptor crucial for dopaminergic neuronal function, thereby driving circadian TH expression through a target-dependent antagonistic mechanism. In conclusion, we identified a molecular connection between the circadian timing system and mood regulation, suggesting that REV-ERBα could be targeting in the treatment of circadian rhythm-related affective disorders.

A novel intracellular role of matrix metalloproteinase-3 during apoptosis of dopaminergic cells

We have previously demonstrated that the active form of matrix metalloproteinase‐3 (actMMP‐3) is released from dopamine(DA)rgic neurons undergoing apoptosis. Herein, whether actMMP‐3 might be generated intracellularly, and if so, whether it is involved in apoptosis of DArgic neurons itself was investigated in primary cultured DArgic neurons of wild‐type, MMP‐3 knockout animals, and CATH.a cells. During apoptosis, gene expression of MMP‐3 is induced, specifically among the various classes of MMPs, generating the proform (55 kDa) which is subsequently cleaved to the catalytically active actMMP‐3 (48 kDa) involving a serine protease. Intracellular actMMP‐3 activity is directly linked to apoptotic signaling in DArgic cells: (i) Pharmacologic inhibition of enzymatic activity, repression of gene expression by siRNA, and gene deficiency all lead to protection; (ii) pharmacologic inhibition causes attenuation of DNA fragmentation and caspase 3 activation, the indices of apoptosis; and (iii) inhibition of the pro‐apoptotic enzyme c‐Jun N‐terminal protein kinase leads to repression of MMP‐3 induction. Under the cell stress condition, MMP‐3 is released as actMMP‐3 rather than the proform (proMMP‐3), and catalytically active MMP‐3 added to the medium does not cause cell death. Thus, actMMP‐3 seems to have a novel intracellular role in apoptotic DArgic cells and this finding provides an insight into the pathogenesis of Parkinson’s disease.

Bromocriptine activates NQO1 via Nrf2-PI3K/Akt signaling: Novel cytoprotective mechanism against oxidative damage

Parkinsons disease (PD) is a neurodegenerative disorder associated with selective loss of dopaminergic neurons in the substantia nigra. Because oxidative stress caused by dopamine oxidation to dopamine quinone is suggested as a major factor contributing to the pathogenesis of PD, the induction of the enzyme that catalyzes the reduction of quinones, NAD(P)H quinone oxidoreductase1 (NQO1), could be a desirable therapeutic strategy to protect cells from oxidative damage. The dopamine agonist bromocriptine is used clinically for PD therapy. In addition to ameliorating the motor deficit via dopamine D2 receptor activation, bromocriptine also has neuroprotective and antioxidative activity. In the present study, we show that bromocriptine upregulates the expression and activity of NQO1, attenuates the increase in the protein-bound quinone in H(2)O(2)-treated PC12 cells, and protects PC12 cells against oxidative damage. Bromocriptine increases the expression and nuclear translocation of a basic leucine zipper transcription factor, nuclear factor-E2-related factor-2 (Nrf2), which is known to be involved in the regulation of numerous antioxidant enzymes via the antioxidant response element. The Nrf2-related cytoprotective and antioxidative effects of bromocriptine are PI3K/Akt pathway-dependent, and are independent of dopamine receptor activation. The cytoprotective effect of bromocriptine in PC12 cells is not affected by the presence of dopamine D2 antagonist, and the bromocriptine-induced Nrf2-ARE activation and cytoprotection against oxidative stress are observed in both dopamine D2 receptor-expressing A7-D2 and non-expressing A7 cells. Taken together, we investigate the novel cytoprotective effect of bromocriptine involving PI3K- and Nrf2-mediated upregulation of the antioxidant enzyme NQO1.

Protective Effect of Sulforaphane against Dopaminergic Cell Death

Parkinsons disease (PD) is a progressive neurodegenerative disorder with a selective loss of dopaminergic neurons in the substantia nigra. Evidence suggests oxidation of dopamine (DA) to DA quinone and consequent oxidative stress as a major factor contributing to this vulnerability. We have previously observed that exposure to or induction of NAD(P)H:quinone reductase (QR1), the enzyme that catalyzes the reduction of quinone, effectively protects DA cells. Sulforaphane (SF) is a drug identified as a potent inducer of QR1 in various non-neuronal cells. In the present study, we show that SF protects against compounds known to induce DA quinone production (6-hydroxydopamine and tetrahydrobiopterin) in DAergic cell lines CATH.a and SK-N-BE(2)C as well as in mesencephalic DAergic neurons. SF leads to attenuation of the increase in protein-bound quinone in tetrahydrobiopterin-treated cells, but this does not occur in cells that have been depleted of DA, suggesting involvement of DA quinone. SF pretreatment prevents membrane damage, DNA fragmentation, and accumulation of reactive oxygen species. SF causes increases in mRNA levels and enzymatic activity of QR1 in a dose-dependent manner. Taken together, these results indicate that SF causes induction of QR1 gene expression, removal of intracellular DA quinone, and protection against toxicity in DAergic cells. Thus, this major isothiocyanate found in cruciferous vegetables may serve as a potential candidate for development of treatment and/or prevention of PD.

Dopamine‐dependent cytotoxicity of tetrahydrobiopterin: a possible mechanism for selective neurodegeneration in Parkinson's disease

Parkinsons disease is a neurodegenerative disorder associated with selective loss of dopaminergic neurons in the substantia nigra. While the underlying cause of this cell death is poorly understood, oxidative stress is thought to play a role. We have previously shown that tetrahydrobiopterin (BH4), an obligatory co‐factor for tyrosine hydroxylase (TH), exerts selective toxicity on dopamine‐producing cells and that this is prevented by antioxidants. This study shows that BH4‐induced dopaminergic cell death is primarily mediated by dopamine, evidenced by findings that (i) BH4 toxicity is increased in proportion to cellular dopamine content; (ii) non‐dopaminergic cells become susceptible to BH4 upon exposure to dopamine; and (iii) depletion of dopamine attenuates BH4 toxicity in dopamine‐producing cells. BH4 causes lipid peroxidation, suggesting involvement of oxidative stress but the toxicity does not require enzymatic oxidation of dopamine. Instead, it seems to involve formation of quinone product(s) because (i) the cell death is attenuated by exposure to or induction of quinone reductase and (ii) BH4‐treated cells show increased formation of protein‐bound quinones, which is inhibited by thiol antioxidants. These data taken together suggest that the presence of both BH4 and dopamine is important in rendering dopaminergic cells vulnerable and that this involves formation of reactive dopamine quinone products.

Role of matrix metalloproteinase-3 in neurodegeneration.

J. Neurochem. (2011) 116, 22–32.

Discovery of Vinyl Sulfones as a Novel Class of Neuroprotective Agents toward Parkinson’s Disease Therapy

Although the etiology of Parkinsons disease (PD) remains elusive, recent studies suggest that oxidative stress contributes to the cascade leading to dopaminergic (DAergic) neurodegeneration. The Nrf2 signaling is the main pathway responsible for cellular defense system against oxidative stress. Nrf2 is a transcription factor that regulates environmental stress response by inducing expression of antioxidant enzyme genes. We have synthesized novel vinyl sulfone derivatives. They exhibited a broad range of activities in inducing HO-1, whose gene expression is under the control of Nrf2. Among them, compound 12g was confirmed to activate Nrf2 and induce expression of the Nrf2-dependent antioxidant enzymes NQO1, GCLC, GLCM, and HO-1, at both mRNA and protein levels in DAergic neuronal cells. This was accompanied by protection of DAergic neurons in both in vitro and MPTP-induced in vivo models of PD. In addition, compound 12g effectively resulted in attenuation of the PD-associated behavioral deficits in the mouse model.