De Giorgio, Francesca; (2019) Investigating TDP-43 mutations in mouse and developing a knockin humanised TDP-43 mouse model of Amyotrophic Lateral Sclerosis (ALS). Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Amyotrophic lateral sclerosis (ALS) is a motor neuron disorder characterised by degeneration of upper and lower motor neurons, with a life expectancy of ~5 years from the disease onset. The majority of cases are sporadic (sALS) but ~10% are familial (fALS). Accumulation of TAR DNA-binding protein (TDP-43) in the cytoplasm and its depletion from the nucleus are a pathogenic signature characterising most of ALS cases. TDP-43 is a highly conserved RNA/DNA binding protein, mainly located in the nucleus and involved in RNA maturation. Mutations in the TARDBP gene cause ALS and so models of ALS-TDP43 are needed to shed light on the pathomechanisms underlying this disease. To date, we count ~30 mouse models of TDP-43, transgenics and knockins, with none of them showing a comprehensive pathological scenario resembling human ALS. This PhD project aimed to create a knockin humanised TDP-43 mouse, replacing the genomic region of mouse Tardbp, from the ATG until the stop codon, with the corresponding sequence from the human orthologue TARDBP and maintaining the mouse untranslated regions (both 5’ and 3’). The Tardbp mouse locus has been modified via a BAC recombineering and homologous recombination, keeping the human gene under the control of the endogenous mouse promoter to reduce the likelihood of interfering with the correct spatial and temporal control of expression. This mouse we aim to produce should recapitulate the normal human TDP-43 expression in vivo under physiological conditions. In addition, 12-month old knockin mice carrying the TardbpQ331K mutation have been characterised to evaluate the effects of this mutation, which is causative for ALS, on TDP-43 functions. To conclude, using mouse embryonic fibroblasts, collected from mice carrying TDP-43 gain and loss of function mutations in the Tardbp gene, we investigated the correlation between mutant TDP-43 and the changes in alternative splicing of hnRNP A1, regulated by TDP-43, as well as their effect on stress granules formation under oxidative stress conditions.

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