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.

Crif1 deficiency in dopamine neurons triggers early-onset parkinsonism.

Mitochondrial dysfunction has been implicated in Parkinson's Disease (PD) progression; however, the mitochondrial factors underlying the development of PD symptoms remain unclear. One candidate is CR6-interacting factor1 (CRIF1), which controls translation and membrane insertion of 13 mitochondrial proteins involved in oxidative phosphorylation. Here, we found that CRIF1 mRNA and protein expression were significantly reduced in postmortem brains of elderly PD patients compared to normal controls. To evaluate the effect of Crif1 deficiency, we produced mice lacking the Crif1 gene in dopaminergic neurons (DAT-CRIF1-KO mice). From 5 weeks of age, DAT-CRIF1-KO mice began to show decreased dopamine production with progressive neuronal degeneration in the nigral area. At ~10 weeks of age, they developed PD-like behavioral deficits, including gait abnormalities, rigidity, and resting tremor. L-DOPA, a medication used to treat PD, ameliorated these defects at an early stage, although it was ineffective in older mice. Taken together, the observation that CRIF1 expression is reduced in human PD brains and deletion of CRIF1 in dopaminergic neurons leads to early-onset PD with stepwise PD progression support the conclusion that CRIF1-mediated mitochondrial function is important for the survival of dopaminergic neurons.

Optogenetic Inhibition of the Subthalamic Nucleus Reduces Levodopa-Induced Dyskinesias in a Rat Model of Parkinson's Disease.

BACKGROUND: The inhibition of neuronal activity by electrical deep brain stimulation is one of the mechanisms explaining the amelioration of levodopa-induced dyskinesia. However, electrical deep brain stimulation cannot specifically activate or inactivate selected types of neurons. OBJECTIVES: We applied optogenetics as an alternative treatment to deep brain stimulation for levodopa-induced dyskinesia, and also to confirm that the mechanism of levodopa-induced dyskinesia amelioration by subthalamic nucleus deep brain stimulation is mediated through neuronal inhibition. METHODS: 6-hydroxydopamine-induced hemiparkinsonian rats received injections of hSynapsin1-NpHR-YFP adeno-associated virus (AAV) or hSynapsin1-YFP AAV. Two weeks after viral injections, all rats were treated with daily injections of levodopa. Then, the optic fiber was implanted into the ipsilateral subthalamic nucleus. We performed various behavioral tests to evaluate the changes in levodopa-induced dyskinesias after optogenetic expression and illumination in the subthalamic nucleus. RESULTS: The behavioral tests revealed that optical inhibition of the subthalamic nucleus significantly ameliorated levodopa-induced dyskinesia by reducing the duration of the dyskinesias as well as the severity of axial dyskinesia. CONCLUSIONS: These findings will provide a useful foundation for the future development of optogenetic modulation systems that could be considered as an approach to dyskinesia therapy.

Macrophage migration inhibitory factor mediates the antidepressant actions of voluntary exercise.

Voluntary exercise is known to have an antidepressant effect. However, the underlying mechanism for this antidepressant action of exercise remains unclear, and little progress has been made in identifying genes that are directly involved. We have identified macrophage migration inhibitory factor (MIF) by analyzing existing mRNA microarray data and confirmed the augmented expression of selected genes under two experimental conditions: voluntary exercise and electroconvulsive seizure. A proinflammatory cytokine, MIF is expressed in the central nervous system and involved in innate and adaptive immune responses. A recent study reported that MIF is involved in antidepressant-induced hippocampal neurogenesis, but the mechanism remains elusive. In our data, tryptophan hydroxylase 2 (Tph2) and brain-derived neurotrophic factor (Bdnf) expression were induced after MIF treatment in vitro, as well as during both exercise and electroconvulsive seizure in vivo. This increment of Tph2 was accompanied by increases in the levels of total serotonin in vitro. Moreover, the MIF receptor CD74 and the ERK1/2 pathway mediate the MIF-induced Tph2 and Bdnf gene expression as well as serotonin content. Experiments in Mif(-/-) mice revealed depression-like behaviors and a blunted antidepressant effect of exercise, as reflected by changes in Tph2 and Bdnf expression in the forced swim test. In addition, administration of recombinant MIF protein produced antidepressant-like behavior in rats in the forced swim test. Taken together, these results suggest a role of MIF in mediating the antidepressant action of exercise, probably by enhancing serotonin neurotransmission and neurotrophic factor-induced neurogenesis in the brain.

Caspase-9 activation and Apaf-1 cleavage by MMP-3.

We have previously demonstrated that matrix metalloprotease-3 (MMP-3) can act inside the cell to trigger apoptosis in response to various cell stresses in dopaminergic neuronal cells. However, the mechanism by which MMP-3 activity leads to caspase-3 activation in apoptotic signaling was not known. In the present study, we found that MMP-3 acts upstream of caspase-9. Overexpression of wild type MMP-3, but not mutant MMP-3, generated the enzymatically active 35kD caspase-9. The caspase-9 activation was absent in MMP-3 knockout cells, but was present when these cells were transfected with wild type MMP-3 cDNA. It was elevated in cells that were under a MMP-3-inducing ER stress condition, and this was attenuated by pharmacologic inhibition and gene knockdown of MMP-3. Incubation of recombinant catalytic domain of MMP-3 (cMMP-3) with procaspase-9 was not sufficient to cause caspase-9 activation, and an additional cytosolic factor was required. cMMP-3 was found to bind to the cytosolic protein Apaf-1, as determined by changes in surface plasmon resonance, and to cleave Apaf-1. Pharmacological inhibition, knockout, and knockdown of MMP-3 attenuated the cleavage. Taken together, the present study demonstrates that MMP-3 leads to caspase-9 activation and suggests that this occurs indirectly via a cytosolic protein, possibly involving Apaf-1.

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.

Evaluation of a multiple system atrophy model in rats using multitracer microPET.

BACKGROUND: A double toxin-double lesion strategy is appropriate for mimicking of striatonigral degeneration. Because knowledge of human pathology is limited, animal models must be well characterized prior to testing of therapeutic approaches to treat multiple system atrophy. In double-toxin animal models, however, reduced contralateral rotation after apomorphine injection is restored within a few weeks via an unknown mechanism; the animals thus revert to PD status. We assessed this phenomenon using multitracer microPET and tissue staining. METHODS: Five adult male Wistar rats received injections of 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle (MFB), followed 3 weeks later by injections of quinolinic acid (QA) into the ipsilateral striatum. Apomorphine-induced rotation tests were performed 1 week after each injection, and 6 and 10 weeks after QA injection. Rotarod tests were performed weekly after 6-OHDA injection. MSA-p status was characterized by microPET 5 and 10 weeks after QA injection using the tracers 2-deoxy-2-[(18)F]-fluoro-D-glucose ([(18)F]-FDG) and [(18)F]-N-(3-fluoropropyl)-2-carbomethoxy-3-(4-iodophenyl)nortropane ([(18)F]-FP-CIT). Histological changes were evaluated by tyrosine hydroxylase (TH) and cresyl violet staining. RESULTS: The numbers of apomorphine-induced rotations increased contralaterally after 6-OHDA lesions were created, but decreased significantly after QA administration (p = 0.007). Five weeks after QA injection, however, contralateral rotation again increased and persisted for 1 month. Rotarod rotation differed significantly between the intact and PD states (p < 0.05), but not between the PD and MSA-p states. MicroPET revealed glucose hypometabolism and dopamine transporter (DAT) impairment on the lesioned side of the striatum 1 and 2 months after QA lesion surgery. Loss of nigral cells was confirmed by TH immunostaining, and striatal atrophy was observed upon cresyl violet staining. CONCLUSION: Pathological changes consistent with MSA-p can be generated by the double toxin-double lesion method and persist during follow-up. Behavioral tests, such as drug-induced rotation and rotarod tests, are not appropriate for long-term follow-up in the MSA-p model, suggesting the need for development of more appropriate behavioral tests.

Pyruvate Prevents Dopaminergic Neurodegeneration and Motor Deficits in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the selective loss of dopamine(DA)rgic neurons in the substantia nigra of the midbrain, and primarily causes motor symptoms. While the pathological cause of PD remains uncertain, oxidative damage, neuroinflammation, and energy metabolic perturbation have been implicated. Pyruvate has been shown neuroprotective in animal models for many neurological disorders, presumably owing to its potent anti-oxidative, anti-inflammatory, and energy metabolic properties. We therefore investigated whether exogenous pyruvate could also protect nigral DA neurons from degeneration and reverse the associated motor deficits in an animal model of PD using the DA neuron-specific toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP (20 mg/kg) was injected four times every 2 h into the peritoneum of mice, which resulted in a massive loss of DA neurons as well as an increase in neuronal death and cytosolic labile zinc overload. There were rises in inflammatory and oxidative responses, a drop in the striatal DA level, and the emergence of PD-related motor deficits. In comparison, when sodium pyruvate was administered intraperitoneally at a daily dose of 250 mg/kg for 7 days starting 2 h after the final MPTP treatment, significant relief in the MPTP-induced neuropathology, neurodegeneration, DA depletion, and motor symptoms was observed. Equiosmolar dose of NaCl had no neuroprotective effect, and lower doses of sodium pyruvate did not have any statistically significant effects. These findings suggest that pyruvate has therapeutic potential for the treatment of PD and related neurodegenerative diseases.

A Novel Pyrazolo[3,4-d]pyrimidine Induces Heme Oxygenase-1 and Exerts Anti-Inflammatory and Neuroprotective Effects.

The anti-oxidant enzyme heme oxygenase-1 (HO-1) is known to exert anti-inflammatory effects. From a library of pyrazolo[3,4-d]pyrimidines, we identified a novel compound KKC080096 that upregulated HO-1 at the mRNA and protein levels in microglial BV-2 cells. KKC080096 exhibited anti-inflammatory effects via suppressing nitric oxide, interleukin-1ß (IL-1ß), and iNOS production in lipopolysaccharide (LPS)-challenged cells. It inhibited the phosphorylation of IKK and MAP kinases (p38, JNK, ERK), which trigger inflammatory signaling, and whose activities are inhibited by HO-1. Further, KKC080096 upregulated anti-inflammatory marker (Arg1, YM1, CD206, IL-10, transforming growth factor-ß [TGF-ß]) expression. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, KKC080096 lowered microglial activation, protected the nigral dopaminergic neurons, and nigral damage-associated motor deficits. Next, we elucidated the mechanisms by which KKC080096 upregulated HO-1. KKC080096 induced the phosphorylation of AMPK and its known upstream kinases LKB1 and CaMKKbeta, and pharmacological inhibition of AMPK activity reduced the effects of KKC080096 on HO-1 expression and LPS-induced NO generation, suggesting that KKC080096-induced HO-1 upregulation involves LKB1/AMPK and CaMKKbeta/AMPK pathway activation. Further, KKC080096 caused an increase in cellular Nrf2 level, bound to Keap1 (Nrf2 inhibitor protein) with high affinity, and blocked Keap1-Nrf2 interaction. This Nrf2 activation resulted in concurrent induction of HO-1 and other Nrf2-targeted antioxidant enzymes in BV-2 and in dopaminergic CATH.a cells. These results indicate that KKC080096 is a potential therapeutic for oxidative stress- and inflammation-related neurodegenerative disorders such as 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.

Role of matrix metalloproteinase-3 in neurodegeneration.

Matrix metalloproteinase-3 (MMP-3) is a member of the class of zinc-dependent proteases known to degrade the extracellular matrix. MMP-3 activity is regulated at three different levels: gene expression, proteolytic activation of the zymogen, and inhibition by the endogenous tissue inhibitors of metalloproteinase. A line of evidence indicates a role of MMP-3 in neurodegeneration. In neuronal cells, MMP-3 expression is increased in response to cell stress, and the cleaved, active MMP-3 participates in apoptotic signaling. In the extracellular space, MMP-3 triggers microglia to produce proinflammatory and cytotoxic molecules as well as MMP-3, which in turn contribute to neuronal damage. MMP-3 is increased in various experimental models of Parkinson's disease that are produced by selective toxins and by inflammagen, and the neuronal death is attenuated by various ways that inhibit MMP-3. α-Synuclein, whose gene mutations are associated with familial forms of Parkinson's disease, is proteolyzed by MMP-3. Contribution of MMP-3 toward the pathogenesis of Alzheimer's disease and other neurodegenerative diseases has also been suggested. Thus, modulation of MMP-3 expression and/or activity could be of therapeutic value for neurodegenerative diseases.

A novel pyrazolo [3,4-d] pyrimidine, KKC080106, activates the Nrf2 pathway and protects nigral dopaminergic neurons.

The transcription factor nuclear factor-erythroid 2-related factor-2 (Nrf2) is known to induce neuroprotective and anti-inflammatory effects and is considered to be an excellent molecular target for drugs related to neurodegenerative disease therapy. Nrf2 activators previously tested in clinical trials were electrophilic, causing adverse effects due to non-selective and covalent modification of cellular thiols. In order to circumvent this issue, we constructed and screened a chemical library consisting of 241 pyrazolo [3,4-d] pyrimidine derivatives and discovered a novel, non-electrophilic compound: 1-benzyl-6-(methylthio)-N-(1-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine (KKC080106). KKC080106 was able to activate Nrf2 signaling as it increases the cellular levels of Nrf2, binds to the Nrf2 inhibitor protein Keap1, and causes the accumulation of nuclear Nrf2. We also observed an increase in the expression levels of Nrf2-dependent genes for antioxidative/neuroprotective enzymes in dopaminergic neuronal cells. In addition, in lipopolysaccharide-activated microglia, KKC080106 suppressed the generation of the proinflammatory markers, such as IL-1ß, TNF-α, cyclooxygenase-2, inducible nitric oxide synthase, and nitric oxide, and inhibited the phosphorylation of kinases known to be involved in inflammatory signaling, such as IκB kinase, p38, JNK, and ERK. As a drug, KKC080106 exhibited excellent stability against plasma enzymes and a good safety profile, evidenced by no mortality after the administration of 2000 mg/kg body weight, and minimal inhibition of the hERG channel activity. Pharmacokinetic analysis revealed that KKC080106 has good bioavailability and enters the brain after oral and intravenous administration, in both rats and mice. In MPTP-treated mice that received KKC080106 orally, the compound blocked microglial activation, protected the nigral dopaminergic neurons from degeneration, and prevented development of the dopamine deficiency-related motor deficits. These results suggest that KKC080106 has therapeutic potential for neurodegenerative disorders such as Parkinson's disease.

Aberrant Tonic Inhibition of Dopaminergic Neuronal Activity Causes Motor Symptoms in Animal Models of Parkinson's Disease.

Current pharmacological treatments for Parkinson's disease (PD) are focused on symptomatic relief, but not on disease modification, based on the strong belief that PD is caused by irreversible dopaminergic neuronal death. Thus, the concept of the presence of dormant dopaminergic neurons and its possibility as the disease-modifying therapeutic target against PD have not been explored. Here we show that optogenetic activation of substantia nigra pars compacta (SNpc) neurons alleviates parkinsonism in acute PD animal models by recovering tyrosine hydroxylase (TH) from the TH-negative dormant dopaminergic neurons, some of which still express DOPA decarboxylase (DDC). The TH loss depends on reduced dopaminergic neuronal firing under aberrant tonic inhibition, which is attributed to excessive astrocytic GABA. Blocking the astrocytic GABA synthesis recapitulates the therapeutic effect of optogenetic activation. Consistently, SNpc of postmortem PD patients shows a significant population of TH-negative/DDC-positive dormant neurons surrounded by numerous GABA-positive astrocytes. We propose that disinhibiting dormant dopaminergic neurons by blocking excessive astrocytic GABA could be an effective therapeutic strategy against PD.

Protective effect of bromocriptine against BH4-induced Cath.a cell death involving up-regulation of antioxidant enzymes.

Previously, we suggested that tetrahydrobiopterin (BH4), an obligatory cofactor for dopamine synthesis, as an intrinsic contributor to dopaminergic neuron vulnerability. The BH4 toxicity is observed in dopamine-producing cells, including Cath.a cells, but not in non-dopaminergic cells. Furthermore, the dopaminergic cell death induced by BH4 is apoptotic in nature and involves oxidative stress, similar to that observed in Parkinson's disease. Accordingly, various antioxidants have been found to protect dopaminergic cells from BH4. This study was undertaken to evaluate protective effects of the dopamine receptor agonist bromocriptine on BH4-induced Cath.a cell death, because bromocriptine has been reported to be an antioxidant with a neuroprotective activity. In the presence of bromocriptine, the increase in LDH activity and mitochondrial cytochrome c release induced by BH4 were significantly abolished. This cytoprotective effect was phosphatidylinositol 3-kinase (PI3K)/Akt pathway-dependent. In addition, bromocriptine was found to up-regulate the expressions of nuclear factor-E2-related factor-2 and antioxidant enzymes including NAD(P)H quinone oxidoreductase 1. Our findings show that bromocriptine stimulates antioxidant defense mechanisms in Cath.a cells and suggest a potential use of bromocriptine as a neuroprotectant.

KMS99220 Exerts Anti-Inflammatory Effects, Activates the Nrf2 Signaling and Interferes with IKK, JNK and p38 MAPK via HO-1.

Neuroinflammation is an important contributor to the pathogenesis of neurodegenerative disorders including Parkinson's disease (PD). We previously reported that our novel synthetic compound KMS99220 has a good pharmacokinetic profile, enters the brain, exerts neuroprotective effect, and inhibits NFκB activation. To further assess the utility of KMS99220 as a potential therapeutic agent for PD, we tested whether KMS99220 exerts an anti-inflammatory effect in vivo and examined the molecular mechanism mediating this phenomenon. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, oral administration of KMS99220 attenuated microglial activation and decreased the levels of inducible nitric oxide synthase and interleukin 1 beta (IL-1b) in the nigrostriatal system. In lipopolysaccharide (LPS)-challenged BV-2 microglial cells, KMS99220 suppressed the production and expression of IL-1b. In the activated microglia, KMS99220 reduced the phosphorylation of IκB kinase, c-Jun N-terminal kinase, and p38 MAP kinase; this effect was mediated by heme oxygenase-1 (HO-1), as both gene silencing and pharmacological inhibition of HO-1 abolished the effect of KMS99220. KMS99220 induced nuclear translocation of the transcription factor Nrf2 and expression of the Nrf2 target genes including HO-1. Together with our earlier findings, our current results show that KMS99220 may be a potential therapeutic agent for neuroinflammation-related neurodegenerative diseases such as PD.

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.

Okadaic acid increases autophagosomes in rat neurons: implications for Alzheimer's disease.

Autophagosomes are accumulated in Alzheimer's disease (AD), but the regulatory pathway of autophagy in AD remains largely unknown. By using electron microscopy, Western blotting, and immunocytochemistry, here we show that autophagosomes are accumulated in rat neurons by okadaic acid (OA), a protein phosphatase-2A inhibitor known to enhance tau phosphorylation, beta-amyloid (Abeta) deposition, and neuronal death, which are the pathological hallmarks of AD. Autophagy can be generally induced via several distinct pathways, such as inhibition of mTOR or activation of beclin-1. Interestingly, OA increased both mTOR and beclin-1 pathways simultaneously, which suggests that autophagy in OA-treated neurons is induced mainly via the beclin-1 pathway, and less so via mTOR inhibition. Finally, inhibition of autophagy by 3MA reduced cytotoxicity in OA-treated neurons. Our novel findings provide new insights into the pathology of and therapeutic intervention for AD.

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 (gp91(phox-/-)), 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.

Cyclooxygenase-2 is involved in oxidative damage and alpha-synuclein accumulation in dopaminergic cells.

Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the formation of prostaglandins from arachidonic acid. The inducible isoform COX-2 is upregulated in the dopaminergic neurons of the substantia nigra of postmortem Parkinson's disease (PD) patients and in neurotoxin-induced Parkinsonism models. COX-2 has attracted significant attention as an important source of oxidative stress in dopaminergic neurons due to its potential to oxidize catechols including dopamine. However, the role of COX-2 in the pathogenesis of PD has not been fully evaluated. Here, we show that COX-2 induces dopamine oxidation, as evidenced by the findings that COX-2 can facilitate dopamine oxidation in a cell-free system and in COX-2-overexpressing SH-SY5Y cells, and that this can be completely abolished by the selective COX-2 inhibitor meloxicam. Increased COX-2 expression causes oxidative protein modification and alpha-synuclein accumulation in dopaminergic cells. These data suggest that an abnormal increase in COX-2 expression causes dopamine oxidation and contributes to the preferential vulnerability of dopaminergic cells as in PD.