title: Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes.
abstract: OBJECTIVE
  To investigate the effects of rapamycin on glutamate uptake in cultured rat astrocytes expressing N-terminal 552 residues of mutant huntingtin (Htt-552).
  
  
  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. [(3)H]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 [(3)H]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 [(3)H]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 [(3)H]glutamate uptake in the astrocytes.
  
  
  CONCLUSIONS
  Rapamcin, an autophagy stimulator, can prevent the suppression of GLT-1 expression and glutamate uptake by mutant Htt-552 in cultured astrocytes.
authors.affiliation: Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science, Suzhou, China.
authors.forename: Lei-lei
authors.initials: LL
authors.lastname: Chen
authors.forename: Jun-chao
authors.initials: JC
authors.lastname: Wu
authors.forename: Lin-hui
authors.initials: LH
authors.lastname: Wang
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Zheng-hong
authors.initials: ZH
authors.lastname: Qin
authors.forename: Marian
authors.initials: M
authors.lastname: Difiglia
authors.forename: Fang
authors.initials: F
authors.lastname: Lin
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D027342:Excitatory Amino Acid Transporter 2 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020123:Sirolimus / Q000494:pharmacology*
chemical_list: D027342:Excitatory Amino Acid Transporter 2; C499524:Htt protein, rat; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D020123:Sirolimus