Investigating the role of FUS, TDP-43 and DYNC1H1 mutations in the etiology of adult and childhood-onset motor neuron disease

Green, Ryan Liam (2017) Investigating the role of FUS, TDP-43 and DYNC1H1 mutations in the etiology of adult and childhood-onset motor neuron disease. Doctoral thesis (PhD), University of Sussex.

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Abstract

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy with lower extremity predominance (SMA-LED) represent two distinctive types of motor neuron disease, the first of which ALS manifests usually as an adult-onset, rapidly progressive, terminal neurodegenerative disorder. Conversely, SMA-LED a childhood-onset disease is mainly characterised by lower limb wasting and intermittently compounded by intellectual disability. Firstly, this thesis represents investigations in the roles of both Fused in Sarcoma (FUS) and TAR-DNA binding protein (TDP-43), two mutated proteins in ALS. Secondly, this thesis explores the implications of SMA-LED mutations in the cytoplasmic dynein heavy chain (DYNC1H1) to investigate disease pathogenesis.

Mutations found in RNA binding proteins FUS and TDP-43 are pathogenic in the adult-onset form of motor neuron disease ALS. These proteins are known to interact with DNA and it is suggested in the literature that FUS in particular has a protective role to repair DNA after lesions. FUS was shown to localise to sub-nuclear regions of UVA induced oxidative damage as well as nucleoli foci after induction of DNA single strand breaks through RNA polymerase II inhibition in mitotic cells and neurons. This re-localisation was inhibited by both caffeine and dipyridamole indicating FUS recruitment via phophodiesterases (PDEs). TDP-43 was found to vacate regions of oxidative damage and showed neuronal specific re-localisation to nucleoli in response to polymerase II inhibition that was not observed in mitotic cells.

These data for the first time show that both FUS and TDP-43 re-localise after DNA damage events. FUS is shown to respond to transcriptional stress perhaps to protect nascent RNA, whereas the expulsion of TDP-43 may indicate a role in transcription coupled excision repair or heterochromatin remodelling.

The SMA-LED autosomal dominant DYNC1H1 mutation p.R399G causes lower limb weakness and muscle atrophy as well as cognitive impairment but the pathogenesis remains unknown. p.R399G mutant fibroblasts exhibit a fragmented Golgi apparatus phenotype rescuable by HDAC6 inhibition in conjunction with a reduced localisation of the dynein complex to the Golgi membranes. Furthermore, western blot and immunoprecipitation assays showed decreased acetylation in mutant fibroblasts as well as an increased interaction between dynein and golgin160 in p.R399G mutants. These data for the first time show that mutations in dynein can modulate acetylation of microtubules and caffect dynein recruitment to the Golgi apparatus. This suggests a novel mechanism in which perturbed dynein-mediated regulation of microtubule acetylation and dynein-Golgi interaction underpin SMA-LED.

Finally, the Dync1h1 mutant Arl SMA-LED mouse model recapitulates phenotypes of the human disease and presents with motor phenotypes. However, this mouse model is yet to be fully characterised and the extent of any aberrant brain development is unknown. Analysis of the model indicated defective cortical organisation in addition to defective cell migration seen in Arl/+ fibroblasts. Furthermore, collapse of the third ventricle and condensation of the corpus callosum was also observed in this model.

These data show that the Arl p.Trp1206Arg mutation in the tail domain of Dync1h1 results in defective cellular migration as well as gross morphological brain deficits that mirror malformation of cortical development (MCD) in patients with SMA-LED. This is likely related to microtubule instability, Golgi abnormalities or impaired force production of dynein leading to suppressed cell migration and neuronal arborisation.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Neuroscience
Subjects: R Medicine > RC Internal medicine > RC0321 Neurosciences. Biological psychiatry. Neuropsychiatry
Depositing User: Library Cataloguing
Date Deposited: 08 May 2017 09:55
Last Modified: 16 Jun 2020 08:01
URI: http://sro.sussex.ac.uk/id/eprint/67574

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