CAG·CTG repeat instability in cultured human astrocytes

Abstract

Cells of the central nervous system (CNS) are prone to the devastating consequences of trinucleotide repeat (TNR) expansion. Some CNS cells, including astrocytes, show substantial TNR instability in affected individuals. Since astrocyte enrichment occurs in brain regions sensitive to neurodegeneration and somatic TNR instability, immortalized SVG-A astrocytes were used as an ex vivo model to mimic TNR mutagenesis. Cultured astrocytes produced frequent (up to 2%) CAG·CTG contractions in a sequence-specific fashion, and an apparent threshold for instability was observed between 25 and 33 repeats. These results suggest that cultured astrocytes recapitulate key features of TNR mutagenesis. Furthermore, contractions were influenced by DNA replication through the repeat, suggesting that instability can arise by replication-based mechanisms in these cells. This is a crucial mechanistic point, since astrocytes in the CNS retain proliferative capacity throughout life and could be vulnerable to replication-mediated TNR instability. The presence of interruptions led to smaller but more frequent contractions, compared to a pure repeat, and the interruptions were sometimes deleted to form a perfect tract. In summary, we suggest that CAG·CTG repeat instability in cultured astrocytes is dynamic and replication-driven, suggesting that TNR mutagenesis may be influenced by the proliferative capacity of key CNS cells.

Tissue specificity in DNA repair: lessons from trinucleotide repeat instability

“…They come in two flavors: plasmid-borne and chromosomal. Replication-based shuttle vectors carry an expanded repeat within a yeast reporter gene whose activity changes upon a change in TNR length [100] or the plasmid is transformed into bacteria to be amplified and the repeats are excised and run on polyacrylamide gels [101]. They have the advantage of being relatively quick to execute but are limited in the sensitivity of the assay and it is unclear how the chromatin structure on the transfected plasmids reflect the endogenous situation.…”

Features of trinucleotide repeat instability in vivo

“…In cultured embryonic fibroblasts from R6/1 mice, CAG repeats in the human HD transgene remained stable in the absence of DNA repair enzymes such as Msh2 and Ogg1 [119]. New genetic assays have been developed using shuttle vectors containing the promoter-TNR-reporter gene sequences [120]. The vector harbors the SV40 origin and the large T antigen gene allowing portability between primate cell lines [120].…”

“…New genetic assays have been developed using shuttle vectors containing the promoter-TNR-reporter gene sequences [120]. The vector harbors the SV40 origin and the large T antigen gene allowing portability between primate cell lines [120]. When propagated in cultured cells, CAG of 25-33 repeats contract at frequencies as high as 1% in both 293T human cells and in COS-1 monkey cells [120].…”

“…The vector harbors the SV40 origin and the large T antigen gene allowing portability between primate cell lines [120]. When propagated in cultured cells, CAG of 25-33 repeats contract at frequencies as high as 1% in both 293T human cells and in COS-1 monkey cells [120]. Plasmids may replicate faster than the cells themselves, so these data indicate that the rate of replication can play a role in the resulting instability.…”

Dynamic mutations as digital genetic modulators of brain development, function and dysfunction

“…Ref. 35), would also have implications for the role of tandem repeat mutagenesis in brain development. It is likely that many dynamic mutations, either at repeat lengths below the threshold for disease or at loci not linked to any unstable repeat expansion disease, can affect gene expression as well as RNA and protein function in subtle ways, which could nonetheless impact on brain development and function.…”