TDP-43 and alternative splicing in motor neurone disease

Lead Research Organisation: University of Sheffield
Department Name: Medicine and Biomedical Science


Motor neurone disease (MND) is a common, tragic degenerative disease that results in progressive weakness, muscle stiffness and wasting. Death is the usual outcome within 2-3 years of diagnosis caused by the loss of cells (called motor neurones) that signal to muscles when and how to contract.
Microscopically, MND is characterized by the loss of a protein called TDP-43 from the cell nucleus (where genes are encoded by DNA) to an abnormal relocation outside the nucleus. TDP-43 is part of the complex mechanism that manufactures proteins according to the code laid down in DNA. One function of TDP-43 is to control the modification of the RNA molecules that are used as templates to make alternative forms of proteins. These changes alter a proteinā€˜s function and can contribute to the cause of disease.
The project aims to investigate whether a loss of TDP-43 from the cell nucleus results in the manufacture of proteins that may change the vulnerability of motor neurones.
Knowing whether altered protein manufacture occurs is important in understanding how the disease process affects and kills motor neurones. It may inform research into motor neurone death, and facilitate the development of treatments aimed at preserving these vital cells.

Technical Summary

This project will investigate alternative RNA splicing of gene transcripts in motor neurone disease (MND).
Recent studies have demonstrated that MND is characterized by the translocation of the protein TDP-43 from the nucleus, associated with ubiquitylation and accumulated in cytoplasmic inclusions. The hypothesis that loss of normal nuclear activity of TDP-43 is important in the pathogenesis of MND will be investigated.
TDP-43 is known to bind (UG)n repeat sequences in the cystic fibrosis transmembrane conductance receptor (CTFR) gene resulting in skipping of exon 9 through alternative RNA splicing as part of the regulation of gene function. CTFR is expressed in human lower motor neurones (LMN). UG(n) sequences are also present in other genes expressed in spinal cord including those for the human cardiac Na+/Ca++ exchanger 1 (NCX1) and molecules potentially implicated in MND: AMPA glutamate receptor subunit GluR2; brain derived neurotrophic factor.
I will use these genes as candidates to explore the hypothesis that reduced nuclear TDP-43 results in altered RNA splicing causing exon skipping. To do this, immunolabelled neurones with and without nuclear TDP-43 expression will be isolated by laser capture microdissection. Two-step linear amplification of RNA followed by quantitative PCR will be used to compare the proportion of spliced and unspliced transcripts. In addition, a TDP-43 knockdown model will be generated in the NSC34 cell line using a lentiviral vector. RNA splicing will be compared with NSC34 cells with normal TDP-43 expression.
Positive findings from this component of the study will determine whether a change in subcellular TDP-43 localisation has demonstrable functional consequences. Whether this has pathogenic significance depends on which genes are affected. Whilst three genes are included with possible implications for MND, a wider survey of aberrant splicing would also be informative. In the second phase I will use Affymetrix GeneChips to investigate gene expression and splicing across the genome using the same comparison of neurones and cell lines with normal and abnormal nuclear TDP-43. This second component will generate additional molecular candidates of potential importance in MND. There is evidence that TDP-43 is less important in SOD1-related familial MND. The host laboratory has autopsy material from SOD1 patients, and transfected cell lines, which will allow me to include this material as an additional comparison group in both phases.
The project will enable me to acquire first hand knowledge of these modern techniques for investigating human nervous system disease with applicability across the neurodegenerative disorders.


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