High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes.
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.
Wang
Linhui
L
Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Medicine, Suzhou 215123, China.
Lin
Fang
F
Wu
Junchao
J
Qin
Zhenghong
Z
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000235:genetics; D000071058:Huntingtin Protein; D002479:Inclusion Bodies / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014162:Transfection; D019680:Trinucleotide Repeat Expansion / Q000235:genetics
C499524:Htt protein, rat; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins