The effects of TAR DNA binding protein mutations on RNA processing associated with Amyotrophic Lateral Sclerosis

Introduction: Amyotrophic Lateral Sclerosis (ALS) is a devastating, chronic progressive neurodegenerative disorder, characterized by the loss of the upper motor neurons in the motor cortex and the lower motor neurons of the brainstem and spinal cord. This leads to muscle weakness, atrophy and paralysis. Death usually occurs 3-5 years from onset. In familial ALS, mutations in TARDBP, encoding the RNA binding protein TDP-43, cause 5% of cases. TDP-43 is mainly localized in the nucleus and has multiple functions, of which the best characterised is regulation of splicing/alternative splicing of hnRNA. In ALS TDP-43 mislocates to the cytoplasm causing the characteristic protein aggregations. The current work investigates the possible effects of both TARDBP missense mutations and a truncation mutation on RNA processing. This was approached by examining the changes in gene expression in both the cytoplasm and nucleus in fibroblasts derived from familial ALS-TARDBP patients. Hypothesis: The cytoplasmic and nuclear transcriptomic profile from mutant TARDBP fibroblasts will generate different transcriptomic profiles than control fibroblasts and will establish transcripts and pathways dysregulated in the presence of mutations in TARDBP. The objectives were 1) to optimize the separation of nuclear and cytoplasmic RNA from patient and control fibroblasts, 2) to compare the expression profiles of the cytoplasmic and nuclear compartments from control and mutant fibroblasts and 3) to determine the effect of both mutation types on gene expression in fALS. Methodology: Fibroblast cell culture from fALS-TARDBP missense mutation, truncation mutation and controls was performed. In addition, cell fractionation and RNA extraction were performed by two methods, osmotic pressure and Trizol and commercially available kit. Gene expression profiling was achieved using the Human Exon Array 1.0 ST, Human Transcriptome Array 2.0 (HTA) and RNA Sequencing. Findings: The presence of a TARDBP mutation causes change in gene expression in fALS. Cytoplasmic fALS-TARDBP missense mutations were significantly enriched with dysregulated RNA processing genes using both the Human Exon Arrays 1.0 ST and the HTAs while cytoplasmic fALS-TARDBP truncated mutation were enriched with dysregulated angiogenesis using the HTA and dysregulated vesicle mediated transport genes using RNA sequencing. The nuclear fALS-TARDBP missense mutations demonstrated dysregulated RNA splicing using both the Human Exon Arrays 1.0 ST and the HTA while nuclear fALS-TARDBP truncated mutation was mainly enriched with G-protein coupled receptors using the HTAs. Therefore, fALS-TARDBP subtype mutations revealed distinct affected biological processes. Conclusion: The different types of TARDBP mutations assayed here have different effects on gene expression and subsequently on cellular pathways involved in TARDBP-related ALS.