Assessment of an allelic series of mouse TDP43 mutations.
Doctoral thesis, UCL (University College London).
Available under License : See the attached licence file.
A chemical mutagenesis approach has been used to generate novel models carrying point mutations in mouse Tardbp, the gene encoding TDP43. TDP43 is a highly conserved protein, which in humans has been shown to be critical in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). The aims of the work described in this thesis are to assess ten TDP43 point mutations at a behavioural, cellular and molecular level to determine whether they are functional and if they can be used as tools or models to understand TDP43 function and dysfunction in the mouse. A comprehensive behavioural assessment of four mouse mutant lines has been completed to assess for TDP43 mutant phenotypes. Behavioural assessment has encompassed general health assessment through to motor capabilities. Tests regularly applied include SHIRPA, grip strength, rotarod and open field. All mutations have also been assessed in vitro using a cystic fibrosis transmembrane regulator (CFTR) add-back assay. The data presented in this thesis characterises the nonsense mutation Q101X, and a missense mutation K160R, generated in mice. The K160R mutation lies within the first RNA recognition motif of TDP43. Compound mutants (TardbpQ101X/K160R) were also generated and assessed. Q101X causes homozygous lethality as occurs in knockout TDP43 mice. This supports the Q101X mutation as being an alternative null allele. Q101X, K160R and compound mutants (TardbpQ101X/K160R) show common neuronal associated phenotypes in the SHIRPA, including limb grasping and a softer body tone. The compound mutants (TardbpQ101X/K160R) also present novel alterations at six months as demonstrated using electron microscopy. Additionally, weight and preliminary anxiety phenotypes have been shown. Q101X heterozygotes were also crossed to transgenic SOD1G93A mice to generate double mutants. Mutations in SOD1 also cause ALS. Modifying effects were assessed through behavioural characterisation with no overt interaction identified. All missense mutations were assessed in vitro using a CFTR mini-gene assay. Out of ten identified point mutations, F210I, a mutation in the second RNA recognition motif of TDP43 showed deficient splicing activity. This was further confirmed ex vivo in mouse embryonic fibroblasts (MEFs). The F210I mutation is an RNA-binding hypomorphic allele. It shows disrupted Tardbp transcript regulation, supporting potential perturbations in TDP43 auto-regulation. There are also further alterations in RNA metabolism, two processes potentially critical to ALS and FTLD-TDP. F210I demonstrates the importance of the second RNA recognition motif in RNA-binding function.
|Title:||Assessment of an allelic series of mouse TDP43 mutations|
|Open access status:||An open access version is available from UCL Discovery|
|Additional information:||Copyright restricted material has been removed from the e-thesis|
|UCL classification:||UCL > School of Life and Medical Sciences
UCL > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Neurology
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