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The Drosophila pericentrin-like protein has been shown to be essential for the formation of the sensory cilia of chemosensory and mechanosensory neurons by mutant analysis in flies, while the in vivo function of pericentrin, a well-studied mammalian centrosomal protein related to microcephalic primordial dwarfism, has been unclear. To determine whether pericentrin is required for ciliogenesis in mammals, we generated and analyzed mice with a hypomorphic mutation of Pcnt encoding the mouse pericentrin. Immunofluorescence analysis demonstrated that olfactory cilia of chemosensory neurons in the nasal olfactory epithelium were malformed in the homozygous mutant mice. On the other hand, the assembly of motile and primary cilia of non-neuronal epithelial cells and the formation of sperm flagella were not affected in the Pcnt-mutant mice. The defective assembly of olfactory cilia in the mutant was apparent from birth. The mutant animals displayed reduced olfactory performance in agreement with the compromised assembly of olfactory cilia. Our findings suggest that pericentrin is essential for the assembly of chemosensory cilia of olfactory receptor neurons, but it is not globally required for cilia formation in mammals.

Recently, the formation of inclusion bodies in several neurodegenerative diseases, including Parkinson's disease, has been associated with the aggregation of unfolded proteins recruited in the centrosome. We have reported previously that rotenone, an insecticide that is used to produce experimental models of Parkinsonism, induced the aggregation of the α-synuclein protein in the centrosome, and it notably affected the structure and function of this organelle in primary cultures of mesencephalic neurons and astrocytes. However, it is still obscure the mechanisms through which the disorganization and centrosomal dysfunction could induce cell death. In this study the rat neuroblastoma B65 cell line was chronically exposed to rotenone, and then the distribution of the centrosomal protein γ-tubulin was studied by immunocytochemistry and Western blot analyses. Finally, the configuration of mitotic spindles and distribution of the p53 protein was observed in the control and rotenone-treated groups. Rotenone treatment increased the number of cells having centrosome overduplication and multipolar mitotic spindles. In contrast, rotenone induced redistribution of the p53 protein, which was colocalized with the γ-tubulin protein in the perinuclear region of cells having overduplicated centrosomes. In addition, the p53 positive signal was markedly intense in cells containing aberrant chromosome segregation and micronuclei. Our results suggest that centrosome overduplication may play an important role in the redistribution of the p53 protein in rotenone-treated cells, and this could represent an alternative mechanism of rotenone to induce apoptosis in neuronal cells.

To reveal specific gene expression in methamphetamine (METH) -induced dopamine neurotoxicity, temporal characteristics of METH-induced changes in gene expression in dopaminergic neuronal cells were examined using the cDNA array and the differential display method. A number of genes in the class of "trafficking & protein turnover," "metabolic pathways," "transmitters & receptors," and "growth factors, cytokines" were upregulated after the METH treatment in the cDNA array assay. Whereas, some genes related to trafficking & protein turnover and "modulators, effectors & intracellular transducers" were decreased by METH. Some proteins associated with synaptic vesicle transportation indeed up- or downregulated after the METH treatment. These data suggest that the protein trafficking and degradation system is involved in the dopaminergic cell death induced by METH. Furthermore, focusing on inflammatory reactions after METH injection, possible neuroprotective property of nonsteroidal anti-inflammatory drugs were examined against METH-induced neurotoxicity. Coadministration of NSAID with METH significantly attenuated striatal dopamine terminal degeneration and microgliosis induced by METH, suggesting that the protective effects are based on their inhibitory activity on production of cytokines and nitric oxides or their suppressive action against microglia activation.

Manganese as environmental factor is considered to cause parkinsonism and induce endoplasmic reticulum stress-mediated dopaminergic cell death. We examined the effects of manganese on parkin, identified as the gene responsible for familial Parkinson's disease, and the role of parkin in manganese-induced neuronal cell death. Manganese dose-dependently induced cell death of dopaminergic SH-SY5Y and CATH.a cells and cholinergic Neuro-2a cells, and that the former two cell types were more sensitive to manganese toxicity than Neuro-2a cells. Moreover, manganese increased the expression of endoplasmic reticulum stress-associated genes, including parkin, in SH-SY5Y cells and CATH.a cells, but not in Neuro-2a cells. Treatment with manganese resulted in accumulation of parkin protein in SH-SY5Y cells and its redistribution to the perinuclear region, especially aggregated Golgi complex, while in Neuro-2a cells neither expression nor redistribution of parkin was noted. Manganese showed no changes in proteasome activities in either cell. Transient transfection of parkin gene inhibited manganese- or manganese plus dopamine-induced cell death of SH-SY5Y cells, but not of Neuro-2a cells. Our results suggest that the attenuating effects of parkin against manganese- or manganese plus dopamine-induced cell death are dopaminergic cell-specific compensatory reactions associated with its accumulation and redistribution to perinuclear regions but not with proteasome system.

The biosynthesis of dopamine (DA) in catecholaminergic neurons is regulated by tyrosine hydroxylase, which converts tyrosine into 3, 4-dihydroxyphenylalanine (L-DOPA). In melanocytes, tyrosinase catalyzes both the hydroxylation of tyrosine and the consequent oxidation of L-DOPA to form melanin. Although it has been demonstrated that tyrosinase is also expressed in the brain, the physiological role of tyrosinase in the brain is still obscure. In this study, to investigate the role of tyrosinase in catecholaminergic neuronal cells, we examined the effects of tyrosinase inhibition on the viability of CATH.a and SH-SY5Y cells using tyrosinase inhibitors-specifically, phenylthiourea (PTU) and 5-hydroxyindole (5-HI)-and the transfection of antisense tyrosinase cDNA. Both inhibitors significantly reduced the cell viability of CATH.a cells in a dose-dependent manner. PTU also specifically enhanced DA-induced cell death, but 5-HI did not. This discrepancy in cell death is probably due to the inhibitors' different mechanism of action: 5-HI inhibits the hydroxylation of tyrosine as a competitor for the substrate to induce cell death that may be due to depletion of DA, whereas PTU mainly inhibits the enzymatic oxidation of L-DOPA and DA rather than tyrosine hydroxylation to increase consequently autooxidation of DA. Indeed, the intracellular DA content in CATH.a cells was enhanced by PTU exposure. In contrast, PTU showed no enhancing effects on DA-induced cell death of SH-SY5Y cells, which express little tyrosinase. Furthermore, transfection with antisense tyrosinase cDNA into CATH.a cells dramatically reduced cell viability and significantly enhanced DA-induced cell death. These results suggest that tyrosinase controls the intracellular DA content by biosynthesis or enzymatic oxidation of DA, and the dysfunction of this activity induces cell death by elevation of intracellular DA level and consequent gradual autooxidation of DA to generate reactive oxygen species.

Recently, it has been reported that inflammatory processes are associated with the pathophysiology of Alzheimer's disease and that treatment of non-steroidal anti-inflammatory drugs reduce the risk for Alzheimer's disease. In the present study, we examined nitric oxide radical quenching activity of non-steroidal anti-inflammatory drugs and steroidal drugs using our established direct in vitro nitric oxide radical detecting system by electron spin resonance spectrometry. The non-steroidal anti-inflammatory drugs, aspirin, mefenamic acid, indomethacin and ketoprofen directly and dose-dependently scavenged generated nitric oxide radicals. In experiments of nitric oxide radical donor, NOC18-induced neuronal damage, these four non-steroidal drugs significantly prevented the NOC18-induced reduction of cell viability and apoptotic nuclear changes in neuronal cells without affecting the induction of inducible nitric oxide synthase-like immunoreactivity. However, ibuprofen, naproxen or steroidal drugs, which had less or no scavenging effects in vitro, showed almost no protective effects against NOC18-induced cell toxicity. These results suggest that the protective effects of the former four non-steroidal anti-inflammatory drugs against apoptosis might be mainly due to their direct nitric oxide radical scavenging activities in neuronal cells. These direct NO. quenching activities represent novel effects of non-steroidal anti-inflammatory drugs. Our findings identified novel pharmacological mechanisms of these drugs to exert not only their anti-inflammatory, analgesic, antipyretic activities but also neuroprotective activities against neurodegeneration.