System for in vivo ablation of MeCP2-deficient neurons in the Rett Syndrome mouse model

Abstract

Rett syndrome (RTT) is an X-linked neurological disorder caused by loss-of-function mutations in the MECP2 gene. Heterozygous RTT females express a phenotype due to mosaic expression of their wild type (WT) copy of MECP2 caused by random X-chromosome inactivation. Furthermore, neurons expressing WT MECP2, which have the capacity to function normally, are interspersed with neurons expressing the mutant allele, which function inappropriately and compromise the whole neuronal network. Re-expression of MeCP2 in Mecp2-mutant adult mice reverses the neurological phenotype. Thus, therapeutic avenues explored so far to treat RTT, focus on repairing MeCP2 expression in MeCP2-deficient neurons. One of the most challenging aspects of this approach is maintaining MeCP2 levels within physiological limits, as both too much and too little MeCP2 expression lead to neurological disease. An alternative approach based on silencing or removal of the functionally mutant neurons from the mosaic neuronal network has not yet been explored but could in theory be curative. I seek to selectively ablate MeCP2-deficient neurons at different stages in development in the heterozygous mouse model of RTT to determine whether the removal of 50% of neurons in the developing brain is compatible with viability at various developmental stages. The ultimate goal is to ask: does the removal of the MeCP2-deficient neurons ameliorate the RTT-like phenotype?

This thesis describes the development of an inducible in vivo cell ablation system for selective removal of Mecp2 knock-out neurons in the heterozygous RTT mouse model. The novel Mecp2FLExDTR(OFF) genetically engineered mouse line was generated which facilitates Cre-dependent expression of the Diphtheria Toxin Receptor (DTR) from the Mecp2 locus in place of the Mecp2 gene. Mice are resistant to Diphtheria toxin (DT), so expression of the DTR in a cell-type of interest, allows ablation of those cells upon DT administration. Importantly, un-recombined Mecp2FLExDTR(OFF) (cre-negative) mice were shown to be resistant to high doses of DT and therefore comparable to WT littermates. DTR expression was activated only in the presence of Cre recombinase. The combination of the Mecp2FLExDTR(OFF) allele with a transgenic Cre driver allows targeting of DTR expression to MeCP2-deficient cells in the tissue of interest. A preliminary neuronal in vivo ablation experiment was performed where DT was administered to heterozygous Mecp2FLExDTR(OFF)/+,SNAP25-IRES2-cre neonatal mice. At 5 weeks following a single subcutaneous injection of DT at postnatal day 0-2, Mecp2FLExDTR(OFF)/+,cre+ mice showed no signs of overt neurological phenotypes and had no substantial skewing towards MeCP2+ neurons in the brain. It was unclear whether any ablation of MeCP2– neurons had occurred. Further experiments are required to optimise the in vivo ablation protocol to achieve efficient removal of MeCP2– neurons. This thesis discusses the associated challenges and future applications of this inducible in vivo cell ablation mouse model.