High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes

Abstract

Huntington's disease (HD) is caused by an expansion of polyglutamine tract in N-terminus of huntingtin (htt). The mutation of htt leads to dysfunction and premature death of striatal and cortical neurons. However, the effects of htt mutation on glia remain largely unknown. This study aimed to establish a glia HD model using an adenoviral vector to express wild-type and mutant N-terminal huntingtin fragment 1-552 amino acids (htt552) in rat primary cortical astrocytes. We have evaluated optimal conditions for the infection of astrocytes with adenoviral vectors, and the kinetics of the expression of htt552 in astrocytes. The majority of astrocytes expressed the transgene after infection. At 24 h postinfection, the highest rate of infection was 89 + + + + + 3% for the wild-type (htt552-18Q) with a multiplicity of infection (m.o.i.) of 80, and the highest rate of infection was 91 + + + + + 4% for the mutant type (htt552-100Q) with the same viral dose. The duration of expression of htt552 lasted for about 7 days with a relatively high level from 1 to 4 days post-infection. Mutant huntingtin (htt552-100Q) produced the characteristic HD pathology after 3 days by the appearance of cytoplasmic aggregates and intranuclear inclusions. The result of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay showed that the inhibition of viability by virus on astrocytes was also dose-dependent. To obtain high infection rate and low toxicity, the viral dose with an m.o.i. of 40 was optimal to our cell model. The present study demonstrates that adenoviral-mediated expression of mutant htt provides an advantageous system for histological and biochemical analysis of HD pathogenesis in primary cortical astrocyte cultures.

Truncated N-terminal huntingtin fragment with expanded-polyglutamine (htt552-100Q) suppresses brain-derived neurotrophic factor transcription in astrocytes

“…According to our previous study, the percentage of astrocytes bearing cytoplasmic aggregates only reached 15% of htt522-100Q expressing cells in the glial HD model used in the present study [35]. Therefore, besides the influence of aggregates, the soluble htt552-100Q oligomers may also contribute to the decreased transcription of BDNF.…”

“…Moreover, htt552-100Q formed large aggregates in some astrocytes. This recaptured the pathological hallmark of HD [35]. Using this in vitro HD model, we investigated the influence of htt552 on the transcription of BDNF in astrocytes.…”

“…Primary cortical astrocyte cultures Primary astrocytes were purified from 1 to 2 days postnatal Sprague-Dawley rats according to previously described method [35]. In brief, cerebral cortex was isolated, cut into pieces, and collected in ice-cooled phosphate-buffered saline (PBS).…”

Mitochondrial KATP channel involvement in angiotensin II-induced autophagy in vascular smooth muscle cells

“…Immunofluorescent detection of LC3 association with autophagosomes was carried out as previously described [50]. In brief, the VSMCs were fixed with 1:1 methanol and acetone, washed with PBS and then incubated in PBS containing 0.1 % Triton-X-100 for 10 min.…”

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

“…Primary astrocyte cultures were prepared from the cortex of 1-to 2-day-old postnatal SD rat pups as described in a previous study [14] . The ethical committee of Soochow University approved this study.…”