A Study Of The Effects And Mechanisms Of Rotenone On Glutamate Transport System Of Astrocyte In Rats

Rotenone is a naturally occurring chemical obtained from the roots of several plant species belonging to the genus lonchocarpus or Derris. It is widely used in agriculture as a non-specific insecticide since 1940s. But the recent studies indicate rotenone exposure may be associated with neurodegeneration, especially in Parkinson's disease. PD is a late-onset and progressive motor disease marked by selectively nigrostriatal dopaminergic degeneration and lewy body generation. The rats intoxicated with rotenone can induce above-mentioned pathology feature, and appearance symptoms similar to PD. However, the mechanism of rotenone in PD is unclear. Thus, studies in the mechanism of rotenone-induced PD in molecular level is helpful for understanding PD.In recent years, the study on the relationship between excitotoxicity and PD has become the focus. Now generally accepted, glutamate-induced excitotoxicity has been implicated in the etiology of ischemic stroke, trauma as well as neurodegenerative disorders. Some researches show that over-stimulating NMDA receptors by Glu contributes to the death of DA neurons in substantia nigra. Glutamate transporters(GluTs) in astrocytes play an important role in the metabolism of Glu, including uptake and deactivation. Thus, dysfunction of GluTs can aggravate the toxic effects of Glu and damage the neurons in CNS. So we suppose that the selective toxicity of rotenone on DA neurons maybe have a relationship with the disturbance of glutamate transport system in astocytes. The main aim of this study is to elicit the molecular mechanism of rotenone-induced neurodegeneration based on the researches on Glu transporters and metabolism related enzymes.Method: 1.in vivo studies: SD rats were administrated with rotenone for 28 days through subcutaneous injection and Alzet osmotic pump. Histology of striatum, degeneration of neurons and proliferation of astrocytes were observed with HE and immunohistochemistry staining. Glu concentration and Glu transport function were determined with HPLC and isotope labelling. Protein and mRNA expression of GLAST, GLT-1, GS were detected with Western Blot and RT-PCR. 2.in vitro studies: Primary cultured astrocytes were intoxicated with rotenone: Cell morphogy and viability were observed. Protein and mRNA expression of GLAST, GLT-1, GS were detected with Western Blot and RT-PCR. Medical intervention on rotenone-induced neurotoxicity was investigated with PDC, DHK and MSO. Kinetic [Ca2+]i was detected with Ca2+ Imagine and Analysis System.Results:Part one: in vivo studies1. Behavior of the rats was abnormal with some symtoms similar to PD.2. Histology of the rat striatum was changed: the number of neurons declined and the shape of cells changed from polygon to round.3. Positive neurons stained with Fluoro Jade B was found in the striatum of the rats intoxicated with rotenone.4. Numbers of GFAP stained cells were increased, GFAP protein was markedly raised in the striatum of the rats treated with 4.0 mg/kg rotenone.5. Glutamate concentration in rats striatum was increased by 1.2 mg/kg rotenone, that maybe play a role in rotenone neurotoxicity.6. Glutamate transport function in rats striatum was significantly destroyed by rotenone intoxication.7. The expressions of GLAST mRNA and the protein level were decreased significantly in 0.6 mg/kg and 1.2 mg/kg rotenone intoxication groups. It may be the main reason of increased Glu level in rats brain induced by rotenone.8. The expressions of GLT-1 mRNA and the protein level were increased markly in 1.2 mg/kg rotenone intoxication group.9. The increased expression of GS mRNA and GS activity may represent a protective mechanism by brain cells for limiting the neurotoxicity of Glu.Part two: in vitro studies1. Morphology of astrocytes was abnormal, and some cells displayed characters of suspention cells.2. The viability of astrocyte significantly decreased, but astrocytes have higher tolerance to the same dose of rotenone compared with PC12 cells. 3. Cell proliferation was inhibited and the expression of CX43 mRNA was suppressed obviously by 0.5 and 1.0μmol/L rotenone.4. Rotenone induced dysfunction of glutamate uptake in primary cultured astrocytes, that may be responsible for the increased extracellular glutamate concentration.5. The activity of Na+/K+_ATPase in astrocytes was decreased in a dose dependent manner.6. GLAST antibody immunitive cells was decreased. Protein and mRNA expression of GLAST were declined along the increase of rotenone concentration, but numbers of GLT-1 antibody-positive cells increased. Protein and mRNA expression of GLT-1 were raised, that was also associated with the dose of rotenone.7. The expression of GS mRNA and the activity of GS were significantly increased in 1.0 and 2.0μmol/L rotenone intoxication groups.8. Astrocytes pretreated with PDC showed that, the function of glutamate uptake increased obviously compared with rotenone intoxication group, but astrocytes pretreated with DHK showed little change in the function of glutamate uptake.9. Compared with rotenone intoxication group, the function of glutamate uptake of astrocytes pretreated with MSO was increased significantly, indicating an involvement of GS in the excitotoxicity induced by rotenone.10. Rotenone triggered an increase of [Ca2+]i concentration of astrocytes. The trend of the increase of [Ca2+]i was more apparent with high dose of rotenone. TMB-8 could suppress [Ca2+]i in astrocytes. And the increase also could be affected by Ca2+ concentration of cell medium.11. The expression of CaMKⅡαmRNA decreased markly in astocytes intoxicated with rotenone, but p-CaMKⅡexpression was increased obviously, which mean a descent of CaMKⅡactivity.12. When Astrocytes were pretreated with KN-62 to protect CaMKⅡactivity , the function of glutamate uptake was increased obviously compared with rotenone intoxication group, indicating that CaMKⅡmaybe play a important role in glutamate transport of astrocytes.Conclusions:1. Two animal models of rotenone intoxication rats were established through S.C injection and Alzet osmotic pump. In these results suggesting an neuron toxic effect of rotenone on CNS neurons. Morphology of striatum was changed and GFAP protein expression increased, behavior of the rats was abnomal.2. Rotenone induced dysfunction of glutamate transport and high glutamate concentration in rats striatum, which maybe one of the reason to neurodegeneration.3. Dose-dependent toxic effects on primary cultured astrocytes could be induced by rotenone, which decreased significantly the viability of astrocytes, cell proliferation, and CX43 mRNA expression. Astrocytes,however, have higher tolerance to the same dose of rotenone compared with neurons.4. Down-regulation of GLAST induced by rotenone may be responsible for the dysfunction of glutamate uptake in astrocytes in vivo and vitro studies.5. The increased expression of GLT-1 mRNA and protein may represent a protective mechanism by brain cells for limiting the neurotoxicity of Glu in vivo and vitro studies.6. The activity of Na+/K+_ATPase in rats striatum and primary cultured astrocytes decreased in a dose dependent manner.7. The expression of GS mRNA and GS activity were increased significantly in rats striatum and primary cultured astrocytes, that maybe contribute to the protection of neurons from glutamate exicitotoxicity.8. The function of glutamate uptake of astrocytes could be increased obviously by PDC, a non-specific inhibitor of GLAST.9. Astrocytes pretreated with DHK , a specific inhibitor of GLT-1, showed little change in the function of glutamate uptake. Those medical intervention studies suggested that, under the condition of our research, GLAST down-regulation maybe play a lead role in the excitotoxicity of neurons induced by rotenone rather than GLT-1.10. Pretreatment with MSO sigificantly increased the function of glutamate uptake of astrocytes, suggesting an involvement of GS in the excitotoxicity induced by rotenone.11. Ca2+ overloading mainly led by internal flow of extracellular medium could be involved in the mechanism of rotenone neurotoxicity, which would induce glutamate release from astocytes, and aggravate the excitotoxicity of neurons.12. Rotenone affected CaMKⅡexpression on gene and protein level directly, which lead to decreased activity of CaMKⅡ. When astrocytes pretreated with KN-62 to protect CaMKⅡactivity , the function of glutamate uptake increased obviously compared with rotenone intoxication group, it indicated that CaMKⅡmaybe played a important role in glutamate transport of astrocytes.To sum up, rotenone could affect the expression of glutamate transporters and the activity of GS in astrocytes, and lead to high extracellular glutamate concentration, which may be one of the mechanism of its neurotoxicity. Meantime, with Na+/K+_ATPase activity decreasing and Ca2+ overloading, the uptake function of vesicles dependent on ATP and Ca2+ impaired, which influenced the transport of glutamate in astrocytes. These results suggested that how to improve the impaired astrocytes, then enhance the expression and the activity of glutamate transporters and GS, would have great significance to protect neurons in CNS.