Therapeutic testing and epigenetic characterization of Friedreich Ataxia

____________________________________________________________________________ i Acknowledgements ____________________________________________________________________ ii Table of contents _____________________________________________________________________ iii List of figures _______________________________________________________________________ viii List of tables ________________________________________________________________________ xiii List of abbreviations __________________________________________________________________ xv Chapter 1 – Friedreich ataxia: literature review _____________________________________________ 1 1.1 – FXN gene: structure and expression ________________________________________________ 3 1.1.1 – The GAA triplet repeat mutation _______________________________________________ 5 1.1.2 – Instability of GAA expanded repeats ____________________________________________ 6 1.1.3 – Mechanism of expansion of the GAA repeat ______________________________________ 9 1.1.4 – FXN becomes truncated at the transcriptional level _______________________________ 10 1.1. 5 – Genotype/phenotype correlation _____________________________________________ 18 1.2 – Frataxin: structure and function __________________________________________________ 19 1.2.1 – Frataxin is a mitochondrial protein ____________________________________________ 20 1.2.2 – FRDA pathogenesis _________________________________________________________ 21 1.3 – Therapeutic approaches _________________________________________________________ 25 1.3.1 – Removal of excess mitochondrial iron __________________________________________ 25 1.3.2 – Protection from oxidative stress ______________________________________________ 26 1.3.3 – Decrease ATP demand ______________________________________________________ 29 1.3. 4 – Increase of frataxin levels ___________________________________________________ 30 1.4 – FRDA mouse models ____________________________________________________________ 36 1.4. 1 – Knockout mouse models ____________________________________________________ 36 1.4. 2 – Knockin mouse models _____________________________________________________ 37 1.4. 3 – FXN YAC transgenic mouse models ____________________________________________ 38 1.4. 4 – FXN BAC transgenic mouse models ____________________________________________ 60 iv Chapter 2 – General materials and methods _______________________________________________ 61 2.1 – Solutions _____________________________________________________________________ 62 2.2 – Primers ______________________________________________________________________ 64 2.3 – General techniques ____________________________________________________________ 66 2.4 – Genotyping of newborn mice ____________________________________________________ 78 2.5 – Mice breeding for drug treatments ________________________________________________ 79 2.6 – Functional studies during drug treatments __________________________________________ 80 2.7 – Sample collection from mice post drug treatment ____________________________________ 81 2.8 – Real-time PCR/RT-PCR __________________________________________________________ 82 2.9 – Chromatin immunoprecipitation (ChIP) analysis _____________________________________ 83 2.10 – Western blot analysis __________________________________________________________ 85 2.11 – Oxyblot analysis: detection of protein oxidation ____________________________________ 88 2.12 – Southern blot analysis _________________________________________________________ 90 2.13 – Histological analysis of lumbar DRG sections _______________________________________ 92 Chapter 3 – Cannabinoid therapeutic testing in FRDA mice ___________________________________ 95 3.1 – Introduction __________________________________________________________________ 96 3.1.1 – Cannabinoid receptors and ligands ____________________________________________ 97 3.1.2 – CBD and THC as potential therapeutics for FRDA ________________________________ 101 3.2 – Aim of study _________________________________________________________________ 104 3.3 – Materials and methods ________________________________________________________ 105 3.3.1 – Botanical origin of cannabinoids _____________________________________________ 105 3.3.2 – Drug preparation _________________________________________________________ 105 3.3.3 – Study design and drug administration _________________________________________ 106 3.3. 4 – Functional studies during drug treatments _____________________________________ 106 3.3. 5 – Sample collection _________________________________________________________ 107 3.3. 6 – OxyBlot analysis __________________________________________________________ 108 3.3. 7 – Histological analysis _______________________________________________________ 108