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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.

The actin cytoskeleton plays a critical role in the cellular morphological changes. Its organization is essential for neurite extension and synaptogenesis under the processes of neuronal development. Recently, neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP family verprolin-homologous protein (WAVE) have been identified as key molecules, which specifically participate in regulation of actin cytoskeleton through small GTPases. The functions of these factors have been investigated using cultured cells; however, in vivo developmental changes in these factors are not fully understood. In this study, we examined the expression levels and distributions of N-WASP, WAVE and their related proteins in the rat cerebral cortex and hippocampus during postnatal development. Protein levels of these factors were progressively increased during development, and actin was accumulated in membranous fractions. Immunoreactivities for these factors were widely but differentially observed in entire brain. In the developing brain, N-WASP and WAVE seemed to exist in the synapse-rich areas, such as stratum radiatum of hippocampal CA1 subfield. A similar tendency in the distributions of these factors was observed in the mature brain. Taken together, N-WASP, WAVE and their related proteins may participate in normal brain development and synaptic plasticity by regulating the actin cytoskeleton.

A novel pathway of 5-hydroxytryptamine (5-HT)-induced cGMP formation, which does not require Ca 2+ and is distinct from the 5-HT 3 receptor-mediated pathway, is reported to exist in NG108-15 cells. Although the possible involvement of undefined 5-HT receptors and membrane-bound guanylyl cyclase is suggested, the mechanism is not clarified in detail in this Ca 2+ -independent cGMP formation. In the present study, we investigated the activation mechanism of guanylyl cyclase activity. 5-HT-induced Ca 2+ -independent cGMP formation was not observed in the cell membrane preparation. In intact cells, the 5-HT-induced Ca 2+ -independent cGMP formation was inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide (carboxyPTIO), a nitric oxide (NO)-specific trapper, and by 3,7-bis(dimethylamino)-phenothiazinium chloride (methylene blue), a cytosolic guanylyl cyclase-specific inhibitor, suggesting the involvement of NO and cytosolic guanylyl cyclase. Ca 2+ -independent cGMP formation was not inhibited by 1,2-bis( o -aminophenoxy)-ethane- N , N , N ′, N ′-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM), N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and various arginine-derivative nitric oxide synthase (NOS) inhibitors. Our findings suggest that 5-HT stimulation results in the generation of NO followed by cGMP formation in an NOS-independent manner in NG108-15 cells.

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.

We identified a full-length tryptophan hydroxylase (TPH) gene of planarian Dugesia japonica from a head EST database, and named it DjTPH. Based on whole-mount in situ hybridization and immunofluorescence analyses, DjTPH mRNA and protein were mainly expressed in the nervous system, especially ventral nerve cords and eye pigment cells. Furthermore, DjTPH immunoreactivity was clearly detected at commissure axonal connections in the ventral nerve cords. 5-HT was significantly decreased in DjTPH-knockdown planarians compared with control animals. These results suggest that DjTPH is required for 5-HT biosynthesis, and DjTPH antibody is a useful marker for serotonergic neurons in planarians.