Akiko Muroyama

Enhanced susceptibility to MPTP neurotoxicity in magnesium-deficient C57BL/6N mice.

We evaluated the effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57BL/6N mice fed a magnesium (Mg(2+))-deficient diet. On the 3rd week, Mg(2+)-deficient mice displayed increased anxiety- and depression-like behavior. In the Mg(2+)-deficient mice, a low does (10mg/kg) of MPTP treatment decreased dopamine (DA) and its metabolites contents in the striatum, but not in control mice. The same dose of MPTP did not influence these neurochemical markers in the mice fed Mg(2+)-deficient diet for 1 week which did not exhibit the altered emotional behavior. These results indicate that Mg(2+)-deficient mice with altered emotional behavior appear to increase the susceptibility to MPTP neurotoxicity in C57BL/6N mice.

Magnolol Protects against MPTP/MPP(+)-Induced Toxicity via Inhibition of Oxidative Stress in In Vivo and In Vitro Models of Parkinson's Disease.

The aim of this study is to investigate the role of magnolol in preventing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP-) induced neurodegeneration in mice and 1-methyl-4-phenylpyridinium ion-(MPP+-) induced cytotoxicity to human neuroblastoma SH-SY5Y cells and to examine the possible mechanisms. Magnolol (30 mg/kg) was orally administered to C57BL/6N mice once a day for 4 or 5 days either before or after MPTP treatment. Western blot analysis revealed that MPTP injections substantially decreased protein levels of dopamine transporter (DAT) and tyrosine hydroxylase (TH) and increased glial fibrillary acidic protein (GFAP) levels in the striatum. Both treatments with magnolol significantly attenuated MPTP-induced decrease in DAT and TH protein levels in the striatum. However, these treatments did not affect MPTP-induced increase in GFAP levels. Moreover, oral administration of magnolol almost completely prevented MPTP-induced lipid peroxidation in the striatum. In human neuroblastoma SH-SY5Y cells, magnolol significantly attenuated MPP+-induced cytotoxicity and the production of reactive oxygen species. These results suggest that magnolol has protective effects via an antioxidative mechanism in both in vivo and in vitro models of Parkinsons disease.

Loss of striatal dopaminergic terminals during the early stage in response to MPTP injection in C57BL/6 mice

The molecular mechanisms underlying MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced dopaminergic (DAergic) neuronal death in vivo are still not fully understood. To investigate the selective DAergic neurotoxicity, we have developed an immunological technique to isolate DAergic synaptosomes from mouse striatal tissues using an antibody against 20 amino acid residues in the extracellular second loop of dopamine transporter (DAT). The DAT protein level in the isolated DAergic synaptosomes was markedly decreased at 16 h after a single injection of 30 mg/kg MPTP, but not in striatal homogenate and crude synaptosomes fraction. GBR-12909, a dopamine uptake inhibitor, completely reversed the MPTP-induced decrease of DAT protein in the DAergic synaptosomes. These results suggest that the isolated DAergic synaptosomes can be useful to identify mechanisms of loss of the nerve terminals.

Autoantibody-induced internalization of nicotinic acetylcholine receptor α3 subunit exogenously expressed in human embryonic kidney cells.

Autoantibody against nicotinic acetylcholine receptor (nAChR) α3 subunit has been implicated in the pathogenesis of paraneoplastic neurological syndrome. To examine the effect of anti-α3 subunit autoantibody on cell-surface nAChRs, we established human embryonic kidney 293 cells stably co-expressing α3 and β4 subunits. Upon incubation with seropositive patients serum, this cell line showed co-accumulation of patients IgG and α3 subunits in the cytoplasm. These data support the hypothesis that anti-α3 subunit autoantibody induces internalization of cell-surface nAChRs and thereby impairs synaptic transmission.

Oral administration of coenzyme Q10 prevents cytochrome c release from mitochondria induced by 1-methyl-4-phenylpyridinium ion in mouse brain synaptosomes

Coenzyme Q(10) (CoQ(10)) exerts neuroprotective effects in several in vivo and in vitro models of neurodegenerative disorders. However, the mechanisms of action are not fully understood. The aim in this study was to investigate whether oral administration of CoQ(10) could inhibit cytochrome c (cyt c) release from mitochondria induced by 1-methyl-4-phenylpyridinium ion (MPP(+)), which causes dopaminergic cell death by selective inhibition of complex I of the electron transport chain, in mouse brain synaptosomes. An increase of cyt c was detected in the cytosolic fraction from mouse brain synaptosomes treated with MPP(+). Oral administration of CoQ(10) prevented the mitochondrial cyt c release in the MPP(+)-treated synaptosomes. In addition, CoQ(10) did not affect the MPP(+)-induced decrease in mitochondrial oxidation-reduction activity and membrane potential in brain synaptosomes. Our findings demonstrate that MPP(+)-induced mitochondrial cyt c release in brain synaptosomes is prevented by oral administration of CoQ(10) independently of mitochondrial dysfunction prior to the cyt c release.

Oral administration of coenzyme Q10 reduces MPTP-induced loss of dopaminergic nerve terminals in the striatum in mice

The neuroprotective effect of coenzyme Q10 (CoQ10) has been reported in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. In this study, we investigated whether oral administration of CoQ10 could protect the striatal dopaminergic (DAergic) nerve terminals against MPTP-induced toxicity in C57BL/6N mice using immunoisolation technique for DAergic synaptosomes. CoQ10 significantly attenuated decrease in dopamine transporter as well as in synaptophysin and actin protein levels in DAergic synaptosomes from MPTP-treated mice. The effect of CoQ10 was also observed in crude synaptosomes fraction, but not in homogenate. Our results indicate that the nerve terminals are a site for the action of CoQ10 against the MPTP-induced DAergic neurodegeneration.

Neuroprotective effect of coenzyme Q10 at the striatal dopaminergic nerve terminal levels in MPTP-treated C57BL/6N mice

the motor cortex. Moreover, the level of dopamine increase was dependent on the strength of tDCS. The present findings suggest that application of tDCS over the motor cortex may have a direct and/or indirect impact on the monoaminergic systems involving the basal ganglia. The present study is expected to contribute to the future development of non-invasive therapeutic interventions using tDCS for various basal ganglia disorders, such as Parkinson’s disease.

Changes in emotional behavior induced by water immersion restraint stress in mice

The “excitement stage” of the anesthesia is the period following loss of consciousness and marked by excited and delirious activity. During this stage, there may be uncontrolled movements in addition to other complications such as vomiting, breath holding, pupillary dilation, and irregularity of respirations and heart rate. To explore how volatile anesthetics induce uncontrolled movements, we studied the effects of a widely used volatile anesthetic sevoflurane on activities of the striatal neurons with patch-clamp technique using slice preparation taken from mouse striatum. We found that, in the beginning, sevoflurane slightly depolarized and then hyperpolarized the striatal medium spiny projection neurons. The firing pattern induced by current injection changed from regular to irregular during treatment. Evoked IPSCs were suppressed faster and stronger than evoked EPSCs. The frequency of spontaneous IPSCs was significantly diminished whereas that of spontaneous EPSCs were increased transiently but returned to the control level. In the presence of tetrodotoxin, the frequency of miniature IPSCs was diminished significantly but that of miniature EPSCs were unchanged. By contrast, the amplitudes of both spontaneous and miniature IPSCs and EPSCs were unchanged. These results suggest that sevoflurane suppresses the release probability of GABA significantly, and thus transiently excites rather than inhibits the striatal projection neurons, thereby causing involuntary movements.

Early changes in the mitochondrial membrane potential induced by MPP+ in human neuroblastoma SH-SY5Y cells

The neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induces Parkinsonism in human via its metabolite MPP+ (1-methyl-4phenylpyridinium ion). MPP+ is selectively accumulated in dopamine neurons by the dopamine transporter and inhibits complex I of the mitochondrial respiratory chain. Therefore, studies of precise mechanisms of mitochondrial dysfunction are essential to clarify the mechanism of MPTP neurotoxicity. In this study, we investigate that effect of MPP+ on mitochondrial oxidation–reduction (REDOX) activity and the correlation with membrane potential in human neuroblastoma SH-SY5Y cells. The neuroblastoma cells were treated with MPP+ in DMEM containing N2 supplement. Mitochondrial REDOX activity and membrane potential were measured by Alamar blue fluorescence and JC-1 fluorescence ratio, respectively. After 24 h treatment with MPP+ (1–5 mM), mitochondrial REDOX activity was reduced in a concentration-dependent manner, and mitochondrial membrane potential was also decreased. After 4 h treatment with different concentrations of MPP+, mitochondrial REDOX activity was near normal level, whereas decrease of mitochondrial membrane potential was detected. These results indicate that MPP+ can cause a depolarization of mitochondrial membrane potential without early changes in the activity of respiratory chain. Current study is ongoing to elucidate whether the mitochondrial membrane depolarization at an early stage is involved in MPP+ toxicity in the cells.

Effects of different intensity of forced exercise on MPTP-induced neurotoxicity in C57BL/6 mice

It has been reported that low-power laser irradiation (LLI) can modulate various biological processes including cell proliferation. Some reports suggest LLI interferes with cell cycle and inhibits cell proliferation, while others suggest LLI has a stimulatory effect. Mechanism underlying the effect of LLI remains unclear. We have reported that 808 nm (60 mW) LLI has a potential of suppressive effect on the cell proliferation of human-derived glioblastoma A-172. To reveal a wavelength dependency of such an effect, the present studies using 2 other kinds of diode laser generators (405 nm with 27 mW and 532 nm with 60 mW) were designed. The A-172 cells were cultivated in culture dishes or a 96-well plate. No irradiation was applied in control-group, whereas in experimental group, the center of dish or the selected well was irradiated for 20, 40 and 60 min, respectively. Cells were cultivated in CO2 incubator for 1 or 2 days. The dishes or wells were photographed at pre-, just post-irradiation, 24 and 48 hrs after irradiation with a digital camera. Cell countings were performed on the PC screen and the ratio of cell proliferation was measured. MTT colorimetric analysis was also applied at 48 hrs after irradiation and viable cells and cell proliferation were estimated. These analyses showed that 405 nm LLI provided a significant suppressive effect on the proliferation of A-172 cells (p < 0.05 or p < 0.01), and the effect of LLI had a dose-dependency, whereas 532 nm LLI showed a significant stimulatory effect on them (p < 0.05 or p < 0.01), and also had a dose-dependency. Taking the results of 808 nm (60 mW) application into consideration, it is concluded that LLI effects on A-172 cell proliferation have a wavelength dependency.