Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes

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: 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. Conclusion: Rapamcin, an autophagy stimulator, can prevent the suppression of GLT-1 expression and glutamate uptake by mutant Htt-552 in cultured astrocytes.

Primary Cultures of Astrocytes: Their Value in Understanding Astrocytes in Health and Disease

“…3NP was shown to induce an increase in intracellular Ca 2+ in cultured astrocytes which was due to an influx of Ca 2+ by the reverse operation of the Na + -Ca 2+ exchanger, possibly explaining the gliotoxic action of 3NP (225). HD has also been examined in cultured astrocytes infected with an adenovirus carrying the coden gene of N-terminal 552 glutamine repeats (226). In these cells the GLT-1 glutamate transporter and glutamate uptake were found to be significantly reduced.…”

Cellular antiseizure mechanisms of everolimus in pediatric tuberous sclerosis complex, cortical dysplasia, and non–mTOR‐mediated etiologies

“…Another potential mechanism involves direct effects of mTOR inhibitors on astrocytes and GLT‐1 glutamate transporter expression. For example, recent evidence indicates that rapamycin can upregulate GLT‐1 expression …”

Complete but not partial inhibition of glutamate transporters exacerbates cortical excitability in the R6/2 mouse model of Huntington’s disease

“…If GLAST can cope and compensate for the failure of GLT‐1, it might be a possible target for future therapeutic approaches. Interestingly, studies in primary astrocyte cultures from cortex demonstrated that expression of mHtt in astrocytes significantly decreased both mRNA and protein levels of GLT‐1 but not those of GLAST …”