Hyo Jin Son

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.

Matrix metalloproteinase-3 is increased and participates in neuronal apoptotic signaling downstream of caspase-12 during endoplasmic reticulum stress.

Although endoplasmic reticulum (ER) stress-induced apoptosis has been associated with pathogenesis of neurodegenerative diseases, the cellular components involved have not been well delineated. The present study shows that matrix metalloproteinase (MMP)-3 plays a role in the ER stress-induced apoptosis. ER stress induced by brefeldin A (BFA) or tunicamycin (TM) increases gene expression of MMP-3, selectively among various MMP subtypes, and the active form of MMP-3 (actMMP-3) in the brain-derived CATH.a cells. Pharmacological inhibition of enzyme activity, small interference RNA-mediated gene knockdown, and gene knock-out of MMP-3 all provide protection against ER stress. MMP-3 acts downstream of caspase-12, because both pharmacological inhibition and gene knockdown of caspase-12 attenuate the actMMP-3 increase, but inhibition and knock-out of MMP-3 do not alter caspase-12. Furthermore, independently of the increase in the protein level, the catalytic activity of MMP-3 enzyme can be increased via lowering of its endogenous inhibitor protein TIMP-1. Caspase-12 causes liberation of MMP-3 enzyme activity by degrading TIMP-1 that is already bound to actMMP-3. TIMP-1 is decreased in response to ER stress, and TIMP-1 overexpression leads to cell protection and a decrease in MMP-3 activity. Taken together, actMMP-3 protein level and catalytic activity are increased following caspase-12 activation during ER stress, and this in turn plays a role in the downstream apoptotic signaling in neuronal cells. MMP-3 and TIMP-1 may therefore serve as cellular targets for therapy against neurodegenerative diseases.

Doxycycline is Neuroprotective Against Nigral Dopaminergic Degeneration by a Dual Mechanism Involving MMP-3

In Parkinson disease (PD), the dopaminergic (DAergic) neurons in the substantia nigra undergo degeneration. While the exact mechanism for the degeneration is still not completely understood, neuronal apoptosis and inflammation are thought to play roles. We have recently obtained evidence that matrix metalloproteinase (MMP)-3 plays a crucial role in the apoptotic signal in DAergic cells as well as activation of microglia. The present study tested whether doxycycline might modulate MMP-3 and provide neuroprotection of DAergic neurons. Doxycycline effectively suppressed the expression of MMP-3 induced in response to cellular stress in the DAergic CATH.a cells. This was accompanied by protection of CATH.a cells as well as primary cultured mesencephalic DAergic neurons via attenuation of apoptosis. The active form of MMP-3, released under the cell stress condition, was also decreased in the presence of doxycycline. In addition, doxycycline led to downregulation of MMP-3 in microglial BV-2 cells exposed to lipopolysaccharide (LPS). This was accompanied by suppression of production of nitric oxide and TNF-α, as well as gene expression of iNOS, TNF-α, IL-1β, and COX-2. In vivo, doxycycline provided neuroprotection of the nigral DAergic neurons following MPTP treatment, as assessed by tyrosine hydroxylase immunocytochemistry and silver staining, and suppressed microglial activation and astrogliosis as assessed by Iba-1 and GFAP immunochemistry, respectively. Taken together, doxycycline showed neuroprotective effect on DAergic system both in vitro and in vivo and this appeared to derive from anti-apoptotic and anti-inflammatory mechanisms involving downregulation of MMP-3.

Ethyl Pyruvate Rescues Nigrostriatal Dopaminergic Neurons by Regulating Glial Activation in a Mouse Model of Parkinson’s Disease

This study examined whether ethyl pyruvate (EP) 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. Western blotting and immunohistochemistry showed activation of microglial NADPH oxidase and astroglial myeloperoxidase (MPO) and subsequent reactive oxygen species/reactive nitrogen species production and oxidative DNA damage in the MPTP-treated substantia nigra. Treatment with EP prevented degeneration of nigrostriatal DA neurons, increased striatal dopamine levels, and improved motor function. This neuroprotection afforded by EP was associated with the suppression of astroglial MPO expression, NADPH oxidase-, and/or inducible NO synthase-derived reactive oxygen species/reactive nitrogen species production by activated microglia. Interestingly, EP was found to protect DA neurons from 1-methyl-4-phenyl-pyridinium neurotoxicity in cocultures of mesencephalic neurons and microglia but not in neuron-enriched mesencephalic cultures devoid of microglia. The present findings show that EP may inhibit glial-mediated oxidative stress, suggesting that EP may have therapeutic value in the treatment of aspects of Parkinson’s disease related to glia-derived oxidative damage.

Vertical grid test and modified horizontal grid test are sensitive methods for evaluating motor dysfunctions in the MPTP mouse model of Parkinson's disease.

Parkinsons disease (PD) is caused by selective degeneration of the nigral dopaminergic (DArgic) neurons and is accompanied by motor dysfunctions such as tremor, akinesia, and rigidity. Changes in the degree of motor deficit can be utilized as a noninvasive way of assessing alterations in the number of DArgic neurons and/or the amount of DA in animal models of PD, such as mice systemically administrated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study, in order to develop sensitive methods to detect DA-associated motor deficits, we designed a new test called vertical grid test and modified the existing horizontal grid test. After acute MPTP treatment, decreases in the levels of striatal DA (17.4% of control), dihydroxyphenylacetic acid (33.3%), and homovanillic acid (40.5%) were observed. On the modified horizontal grid test, the MPTP-administered mice exhibited average forelimb step distance that was lower than control (82.58%) and correlated with the striatal DA levels. On the vertical grid test, the MPTP-treated mice took dramatically longer total time to climb down (220.94%) and time to make the turn (339.29%) compared to control, and this correlated well with the degree of striatal DA depletion. In comparison, the gait test produced only a small, albeit statistically significant, reduction in the mean stride length (94.55% of control). These results show that the vertical grid test can provide a sensitive measure of motor deficit in mice following administration of MPTP.

A novel compound PTIQ protects the nigral dopaminergic neurones in an animal model of Parkinson's disease induced by MPTP.

BACKGROUND AND PURPOSE In Parkinsons disease, the dopaminergic neurones in the substantia nigra undergo degeneration. While the exact mechanism for the degeneration is not completely understood, neuronal apoptosis and neuroinflammation are thought to be key contributors. We have recently established that MMP‐3 plays crucial roles in dopaminergic cell death and microglial activation.

Cytosolic labile zinc accumulation in degenerating dopaminergic neurons of mouse brain after MPTP treatment.

High levels of labile zinc accumulate in degenerating neurons after brain injury, such as ischemic stroke, trauma, epilepsy, and hypoglycemia. Cytosolic zinc accumulation is also found in brain neurons undergoing apoptosis during development or after neuronal target ablation. Thus, staining with zinc-specific probes can be used to identify neuronal death in the brain. In this study, mice were intraperitoneally given four 20 mg/kg doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at 2-hour intervals, and dopaminergic neurons were then evaluated for zinc accumulation and apoptosis. In the substantia nigra pars compacta, zinc-specific fluorescent dyes revealed that all degenerating neurons, identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), or acid fuchsin or Fluoro-Jade C staining, contained high levels of cytosolic labile zinc. Nuclear condensation/fragmentation was noted in dopaminergic neurons with cytosolic zinc accumulation, indicating apoptotic cell death. These findings support the supposition that cytosolic labile zinc accumulation is an indicator of degenerating dopaminergic neurons in animal models of Parkinsons disease.

A novel compound VSC2 has anti‐inflammatory and antioxidant properties in microglia and in Parkinson's disease animal model

Neuroinflammation through microglial activation is involved in the pathogenesis of neurodegenerative diseases including Parkinsons disease (PD), a major neurodegenerative disorder characterized by dopaminergic neuronal death in the substantia nigra. We examined our novel synthetic compound VSC2 for its anti‐inflammatory properties towards development of a PD therapy.

Induction of NQO1 and Neuroprotection by a Novel Compound KMS04014 in Parkinson's Disease Models

Parkinson’s disease (PD) is a progressive neurodegenerative disorder associated with a selective loss of the neurons containing dopamine (DA) in the substantia nigra pars compacta. Lines of evidence suggest that oxidative stress is a major factor contributing to the vulnerability of DA cells and that the enzyme NAD(P)H quinone oxidoreductase (NQO1) provides protection in these cells. In the present study, we report the synthesis of a novel compound KMS04014 and show that it induces NQO1 gene expression and protects DAergic neuronal cells in both cell culture and animal models of PD. In vitro, KMS04014 increased both mRNA and protein levels of NQO1 and induced nuclear translocation of Nrf2 in the DAergic neuronal cell line CATH.a. It also protected the cells against oxidative stress generated by tetrahydrobiopterin, 1-methyl-4-phenylpyridinium (MPP+), and H2O2. In vivo, KMS04014 attenuated the loss of tyrosine hydroxylase-immunopositive DAergic neurons in the substantia nigra and reduced degeneration of the nigral neurons and striatal fibers in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, an animal model of PD. Taken together, KMS04014 may be utilized toward development of neuroprotective therapy for PD.