Mechanisms of CAG trinucleotide repeat expansion in Huntington's disease – HexpanD

We are using HD mouse models that recapitulate the HD tissue selectivity of CAG/CTG repeats, which allows for correlation analyses (comparing the level of repeat instability in tissues with the level/activity of given factors/pathways). This results in the identification of putative factors contributing to instability. We are focusing our interest on tissues showing both particularly high and low levels of instability, such as the striatum and the cerebellum, respectively. With respect to the BER project part, we have determined at the molar level the concentration of major BER proteins in the striatum and cerebellum of HD mice. The resulting stoichiometries are used in reconstituted repair assays, using purified BER recombinant proteins and oligonucleotide CAG/CTG substrates carrying a DNA lesion. Using this approach, we could specify the role of BER stoichiometry in repair of CAG/CTG repeats and identify critical factors of the instability mechanism. With respect to the epigenetics/transcription project part, we are performing chromatin immunoprecipitation experiments, western-blotting analyses and expression studies using tissues of HD mice to correlate the chromatin structure, the transcriptional activity and the level of instability. The final goal is to identify factors, regulated in a tissue-specific manner and contributing to CAG/CTG instability. Ultimately, such factors might be validated using mouse genetics.

We have shown that BER enzymes are globally more abundant in the cerebellum when compared to the striatum. As a result, repair of CAG/CTG substrates is more efficient using the BER stoichiometry of the cerebellum than that of the striatum. We have shown that the increased efficiency of the cerebellar stoichiometry results from a better processing of the intermediate repaired products. In addition, we have shown that the nature of the sequence and location of the damage modulate repair. They increase or decrease the probability of forming secondary structures, affecting the processing of intermediate repaired products. Collectively, those results suggest that the stoichiometry of BER enzymes may contribute to tissue-selective CAG/CTG instability in HD, increasing the probability of forming slowly processed intermediate products and stable secondary structures, which promotes instability.

BER is DNA repair pathway that removes oxidative DNA damage. Therefore, decreasing oxidative DNA damage might reduce the BER-dependent repeat instability, and the progression of the disease. Moreover, additional DNA repair pathways are involved in CAG/CTG instability, including mismatch repair and nucleotide excision repair. It remains to be determined whether the different DNA repair pathways act in a synergistically or independent manner on the instability of trinucleotide repeat expansions.

Nucleotide sequence, DNA damage location and protein stoichiometry influence base excision repair outcome at disease-associated CAG/CTG repeats. Agathi-Vasiliki Goula 1, Christopher E. Pearson 2,3 Julie Della Maria4, Alan E. Tomkinson 4, David M. Wilson III 5, Karine Merienne 1. Biochemistry, 51(18) :3919-32.

This collaborative work fulfills one objective of the ANR project, which iwas to specify the role of base excision repair in the instability of CAG/CTG repeats. The study could be finalized thanks to the ANR funding.

Disorders associated to trinucleotide repeat (TNR) constitute a unique class of ˜15 neurological, neurodegenerative and neuromuscular genetic diseases. The causing mutations, e.g. the aberrant expansion of TNR, are unstable in germline and somatic cells, leading to further variation of TNR length across successive generations and within tissues. Huntington’s disease (HD), one most frequent TNR disorder, is a fatal disease caused by the expansion of a repetition of CAG triplets within the coding region of the HTT gene (and CTG repeats on the opposite DNA strand). The mutation leads to the production of a protein containing a neurotoxic polyglutamine (polyQ) tract, whose toxicity is proportional to the expansion size. The instability of CAG/CTG repeats has deleterious clinical consequences in HD. First, germline instability underlies the phenomenon of anticipation, whereby the disease worsens over successive generations. Second, somatic instability is tissue selective and most extensive in the striatum, i.e. the brain area that preferentially degenerates. In consequence, highly toxic proteins are produced in this brain region, which accelerates disease progression. Thus, suppressing or limiting CAG/CTG instability in HD cells or tissues could lead to therapeutic benefices. However, this challenging strategy first requires unraveling the complexity of the underlying mechanisms. The models proposed to explain CAG/CTG instability involve formation of stable secondary DNA structures. Mishandling of these aberrant structures by DNA/RNA associated mechanisms, including DNA repair and transcription, can result in CAG/CTG instability. However, the exact contributing mechanisms as well as their potential interplay remains elusive. In addition, epigenetics appears to play a key role in the regulation of TNR instability. However, the underlying mechanism remains fully enigmatic. The aim of the present program is dual. First, we want to address the role of base excision repair (BER) in somatic CAG/CTG instability, thereby extending a study we recently initiated, which suggested that the stoichiometry of BER proteins may contribute to the tissue selectivity of somatic instability in the brain of HD transgenic mice. Using specific reconstituted repair assays, we plan to carefully examine this hypothesis. We intend to assess repair efficacy and quality of oligonucleotide or plasmid substrates with or without CAG/CTG sequences mixed with recombinant BER protein cocktails corresponding to different brain tissue stoichiometries. Second, we want to address the role of epigenetics and transcription in CAG/CTG instability in HD. We want to base our study on three complementary approaches. First, we will compare the chromatin configuration and transcriptional activity at the HD locus of two HD transgenic mouse lines, i.e. the R6/1 and R6/2 lines, which show different degrees of somatic instability. These lines express the same transgene but inserted in different genomic location. They therefore represent excellent models to assess the role of chromatin context in CAG/CTG instability. Second, we will investigate the role of epigenetics in human cells and tissues. In particular, we will search for potential cis-regulatory elements. Third, we will treat HD cells and mice with HDAC inhibitors, which affect chromatin and transcription and could thus have an effect on CAG/CTG instability. The goal is also to evaluate potential side effects of HDAC inhibitors, which are currently used in phase II clinical trials to treat HD patients. We anticipate that the present program provides important insights into the roles and contributions of BER, transcription and epigenetics in CAG/CTG instability in HD.