Pharmacological assessment of methamphetamine-induced behavioral hyperactivity mediated by dopaminergic transmission in planarian Dugesia japonica.

The freshwater planarian Dugesia japonica has a simple central nervous system (CNS) and can regenerate complete organs, even a functional brain. Recent studies demonstrated that there is a great variety of neuronal-related genes, specifically expressed in several domains of the planarian brain. We identified a planarian dat gene, named it D. japonica dopamine transporter (Djdat), and analyzed its expression and function. Both in situ hybridization and immunofluorescence revealed that localization of Djdat mRNA and protein was the same as that of D. japonica tyrosine hydroxylase (DjTH). Although, dopamine (DA) content in Djdat(RNAi) planarians was not altered, Djdat(RNAi) planarians showed increased spontaneous locomotion. The hyperactivity in the Djdat(RNAi) planarians was significantly suppressed by SCH23390 or sulpiride pretreatment, which are D1 or D2 receptor antagonists, respectively. These results suggest that planarians have a Djdat ortholog and the ability to regulate dopaminergic neurotransmission and association with spontaneous locomotion.

Identification of a novel planarian G-protein-coupled receptor that responds to serotonin in Xenopus laevis oocytes.

Planarians are useful animals for regenerative and neuroscience research at the molecular level. Previously, we have reported the distribution and function of neurotransmitter-synthesizing neurons in the planarian central nervous system. In order to understand the neural projections and connections, it is important to understand the distribution of neurotransmitter receptors. In this study, we isolated a serotonin receptor gene and named it DjSER-7 (Dugesia japonica serotonin receptor type 7). DjSER-7-expressing cells were distributed in the planarian brain. According to electrophysiological analysis using Xenopus oocytes, current response was detected upon exposure to serotonin, but not other neurotransmitters in oocytes that were co-injected with mRNAs of both DjSER-7 and Galpha chimera B-2, which can interact with either Gq-, Gs- or Gi-coupled receptor. In contrast, current response was not detected after exposure to neurotransmitters in oocytes injected with only DjSER-7 mRNA. Our results indicated that DjSER-7 responds to serotonin, as indicated by electrophysiological analysis using Xenopus oocytes.

Synergistic effect of galantamine on nicotine-induced neuroprotection in hemiparkinsonian rat model.

Recent studies have reported that smokers tend to be less susceptible to Parkinson's disease (PD) and the stimulation of nicotinic acetylcholine receptor (nAChR) is considered to confer a neuroprotective effect. Galantamine is an acetylcholinesterase inhibitor and an allosteric potentiating ligand for nAChRs. However, the effects of galantamine and nicotine on dopaminergic neurons remain unclear. This study evaluated the neuroprotective effects of galantamine and nicotine in a rat 6-hydroxydopamine (6-OHDA)-induced hemiparkinsonian model. 6-OHDA with or without galantamine and/or nicotine were injected into unilateral substantia nigra of rats. Although methamphetamine-stimulated rotational behavior and dopaminergic neuronal loss induced by 6-OHDA were not inhibited by galantamine alone, those were moderately inhibited by nicotine alone. In addition, 6-OHDA-induced neuronal loss and rotational behavior were synergistically inhibited by co-injection of galantamine and nicotine. These protective effects were abolished by mecamylamine, an nAChR antagonist. We further found that alpha7 nAChR was expressed on both tyrosine hydroxylase (TH)-immunopositive and TH-immunonegative neurons in the SNpc. A combination of galantamine and nicotine greatly suppressed 6-OHDA-induced reduction of TH-immunopositive/alpha7 nAChR-immunopositive neurons. These results suggest that galantamine synergistically enhances the neuroprotective effect of nicotine against 6-OHDA-induced dopaminergic neuronal loss through an allosteric modulation of alpha7 nAChR activation.

Protective effect of planarian DJ-1 against 6-hydroxydopamine-induced neurotoxicity.

DJ-1/PARK7 has multiple functions as an antioxidant, an oncogene, and a molecular chaperone in vertebrates, and loss-of-function mutations in DJ-1 cause early onset of Parkinson's disease. However, the function of invertebrate DJ-1 remains unknown. In order to investigate the function of planarian DJ-1, we isolated the planarian DJ-1 gene Dugesia japonica DJ-1 (DjDJ-1) and analyzed its expression and function. In situ hybridization analysis revealed that DjDJ-1 mRNA was expressed throughout the body, including the central nervous system, cells surrounding the pharynx, and stem cells. Planarian DjDJ-1 protein exhibited antioxidant function, similar to human DJ-1, as evidenced by the fact that recombinant DjDJ-1 protein reduced reactive oxygen species and protected human neuroblastoma SH-SY5Y cells from cell death. In addition, dopaminergic neurons in DjDJ-1(RNAi) planarians became susceptible to 6-hydroxydopamine, a dopaminergic neurotoxin. These results suggest that planarians have a DJ-1 ortholog, which has conserved antioxidant and neuroprotective functions.

Oxidative stress induction of DJ-1 protein in reactive astrocytes scavenges free radicals and reduces cell injury.

Astrocytes, one of the predominant types of glial cells, function as both supportive and metabolic cells for the brain. Under cerebral ischemia/reperfusion-induced oxidative conditions, astrocytes accumulate and activate in the ischemic region. DJ-1 has recently been shown to be a sensor of oxidative stress in living cells. However, the function of astrocytic DJ-1 is still unknown. In the present study, to clarify the effect of astrocytic DJ-1 protein under massive oxidative insult, we used a focal ischemic rat model that had been subjected to middle cerebral artery occlusion (MCAO) and reperfusion. We then investigated changes in the distribution of DJ-1 in astrocytes, DJ-1 release from cultured astrocytes, and the effects of recombinant DJ-1 protein on hydrogen peroxide (H(2)O(2))-induced death in normal and DJ-1-knockdown SH-SY5Y cells and on in vitro scavenging of hydroxyl radicals ((*)OH) by electron spin resonance spectrometry. At 24 h after 2-h MCAO and reperfusion, an infarct lesion was markedly observed using magnetic resonance imaging and 2,3,5-triphenyltetrazolium chloride staining. In addition, reactive astrocytes enhanced DJ-1 expression in the penumbral zone of the ischemic core and that DJ-1 protein was extracellularly released from astrocytes by H2O2 in in vitro primary cultures. Although DJ-1-knockdown SH-SY5Y cells were markedly vulnerable to oxidative stress, treatment with glutathione S-transferase-tagged recombinant human DJ-1 protein (GST-DJ-1) significantly inhibited H(2)O(2)-induced cell death. In addition, GST-DJ-1 protein directly scavenged (*)OH. These results suggest that oxidative stress induces the release of astrocytic DJ-1 protein, which may contribute to astrocyte-mediated neuroprotection.