INVESTIGATING THE IMMEDIATE CONSEQUENCES OF NORMAL AND MUTANT HTT LOSS IN HD-HESC THROUGH THE DTAG SYSTEM.
Huntington’s Disease (HD) is a neurodegenerative disorder cause by a dominant CAG triplet repeat expansion (poly-glutamine(Q) in the protein) in the first exon of the Huntingtin gene (HTT). In the healthy population, the number of CAG repeats range between 9 and 35, while an expansion above 36 CAG repeats causes the manifestation of the pathology later in life. Numerous studies indicate that the pathological CAG length causes the poly-Q expanded protein to acquire a toxic function which ultimately kills the neurons1–10. Other studies show that several disfunctions associated with the presence of the mutant protein can be phenocopied in cells and mice deleted of the healthy gene, suggesting that a loss of function mechanism may also contributes to HD11–13. Thus the disease seems to be the result of a two-component pathological mechanism; first a loss-of-function (LOF) due to reduced normal HTT physiological activity, and second a gain-of-function (GOF) due to mutant HTT toxic effects14. Being able to discriminate between LOF and GOF phenotypes is especially relevant when considering the ongoing gene silencing clinical studies aiming at reducing HTT level either in an allele-specific or non-allele-specific manner15–17. On top of that, transcriptional alterations have been reported both in HD patients and HD mouse models18, and several evidence have linked HTT to gene expression1,19–27. However, these data refer to situations in which a transcriptional balance has already been established following the genetic perturbation. No data is available regarding the mechanisms acutely implemented by the cell immediately after perturbation to re-establish transcriptional balance in response to those changes. For these reasons, in this project we aimed at generating a human embryonic stem cell (hESC) HD model in which normal or mutant HTT can be rapidly and efficiently removed upon exposure to a small molecule. This is made possible by a degradation tag (dTAG)28 that, when fused to HTT, induces a proteosome-mediated degradation upon exposure to a cell permeable ligand. Accordingly, the first part of the project consists in the generation of two dTAG-hESC-HD lines in which either the normal or the mutant allele were targeted by Cas9-assisted genome editing followed by the assessment of the efficiency and degradation kinetic of tagged HTT in both cell lines. These experiments revealed complete tagged HTT loss between one and two hours of treatment with a ligand concentration of 10-7 M. Moreover, no difference was observed when comparing degradation kinetics of normal and mutant HTT in self-renewing dTAG-hESC lines. We aim to use these lines to study the immediate transcriptomic changes and the potential compensatory mechanisms established by the cells in response to normal or mutant HTT protein depletion. Therefore, a first RNA-seq experiment was performed to investigate over time transcriptional changes during dTAG-HTT degradation. This experiment revealed no substantial transcriptomic changes between normal and the HTT-depleted state of self-renewing dTAG-hESC HD lines. We are now looking into the transcriptional changes driven by HTT degradation in hESC in vitro derived neurons that can provide useful information on the biosafety of the ongoing HTT-lowering approaches for HD treatment.
Settore BIO/14 - Farmacologia, Settore BIO/11 - Biologia Molecolare
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