Abstract
Aim:
To investigate the effects of rapamycin on glutamate uptake in cultured rat astrocytes expressing N-terminal 552 residues of mutant huntingtin (Htt-552).
Methods:
Methods: Primary astrocyte cultures were prepared from the cortex of postnatal rat pups. An astrocytes model of Huntington's disease was established using the astrocytes infected with adenovirus carrying coden gene of N-terminal 552 residues of Huntingtin. The protein levels of glutamate transporters GLT-1 and GLAST, the autophagic marker microtubule-associated protein 1A/1B-light chain 3 (LC3) and the autophagy substrate p62 in the astrocytes were examined using Western blotting. The mRNA expression levels of GLT-1 and GLAST in the astrocytes were determined using Real-time PCR. 3H]glutamate uptake by the astrocytes was measured with liquid scintillation counting.
Results:
The expression of mutant Htt-552 in the astrocytes significantly decreased both the mRNA and protein levels of GLT-1 but not those of GLAST. Furthermore, Htt-552 significantly reduced 3H]glutamate uptake by the astrocytes. Treatment with the autophagy inhibitor 3-MA (10 mmol/L) significantly increased the accumulation of mutant Htt-552, and reduced the expression of GLT-1 and 3H]glutamate uptake in the astrocytes. Treatment with the autophagy stimulator rapamycin (0.2 mg/mL) significantly reduced the accumulation of mutant Htt-552, and reversed the changes in GLT-1 expression and 3H]glutamate uptake in the astrocytes.
Conclusion:
Rapamcin, an autophagy stimulator, can prevent the suppression of GLT-1 expression and glutamate uptake by mutant Htt-552 in cultured astrocytes.
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Acknowledgements
We thank Prof Hai-yan LIU at Soochow University for providing help in the measurement of 3H]-glutamate. This work is supported by grants from the National Natural Science Foundation of China (No 30600197).
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Chen, Ll., Wu, Jc., Wang, Lh. et al. Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes. Acta Pharmacol Sin 33, 385–392 (2012). https://doi.org/10.1038/aps.2011.162
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DOI: https://doi.org/10.1038/aps.2011.162
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