Molecular analysis of the motor neuron disease-associated protein, TDP-43 (2013)

Amyotrophic lateral sclerosis (ALS) also known as motor neuron disease is a progressive neurological disorder that is characterized by the degeneration of upper and lower motor neurons. Mutations in TDP-43, an RNA binding and nucleo-cytoplasmic shuttling protein, have been reported to cause ALS in humans and cytoplasmic inclusions containing TDP-43 have been found as a pathological feature of various neurological diseases (e.g., frontotemporal lobar degeneration, ALS, Corticobasal degeneration, Lewy body related diseases and Pick's disease). This thesis aims to identify mRNA targets of TDP-43 in the mammalian central nervous system (CNS) and to find the effect of mutant TDP-43 on mammalian CNS alternative splicing.In the first two result chapters (3.1 and 3.2) of this thesis, we report mRNA targets of TDP-43 in mice CNS by combining RNA immunoprecipitation (RIP) and microarray (chip) techniques. RNA binding proteins such as TDP-43 are known to bind different subsets of mRNA based on tissue type, thereby regulating diverse functions. In chapter 3.1, we report 1839 mRNA transcripts as potential targets of TDP-43 in mouse brain. Pathway analysis of brain data showed that close to 100 targets identified are associated with synaptic function. In chapter 3.2, we report mRNA targets of TDP-43 in mouse spinal cord. This is the first study to report RNA binding partners of TDP-43 in mouse spinal cord. We confirmed SNORD115, a small nucleolar RNA (snoRNA) previously shown to be associated with TDP-43, as a major binding partner in mouse spinal cord. This was however not found in the mouse brain data. SNORD115 is responsible for regulating alternative splicing and other non-coding RNAs. We also report several other mRNA transcripts identified as TDP-43 binding partners in mouse spinal cord in chapter 3.2.Chapter 3.3 was mainly carried out to identify mRNA binding targets of TDP-43 in a human cell line of neuronal origin. RIP-chip analysis was carried out on primary neurospheres derived from olfactory epithelial cells of an ALS patient carrying a mutation in C-terminal region of TDP-43 (M337V) and a healthy individual.In addition to examining mRNA targets of TDP-43, in chapter 3.4, we looked at the effect of mutant TDP-43 on alternative splicing in mammalian CNS. Splicing is a natural mechanism by which eukaryotes remove introns to produce a mature transcript. Alternative splicing (AS) is a process by which a variety of mature transcripts (isoforms) are made from the same pre-mRNA transcript resulting in a variety of isoforms of the proteins they encode. Abnormal AS has been reported in various neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and sporadic Alzheimer disease. TDP-43 is known to regulate pre-mRNA splicing, RNA transport and stability. As reported in chapter 3.1 and 3.2, in the central nervous system of rodents (i.e., mice) TDP-43 binds to various mRNA targets. In chapter 3.4, we have utilised a transgenic mouse that overexpresses human TARDBP, the gene encoding TDP-43 that contains an ALS causing mutation (A315T). These mice develop motor neuron disease with symptoms similar to humans. RNA from wild type and transgenic mouse brain and spinal cord tissue was hybridized to exon microarrays to elucidate the global effect of mutated TDP-43 on AS in the CNS of transgenic mice. Microarray results were verified in selected genes by PCR and qPCR. Pre-mRNA splicing of various transcripts, previously identified as RNA binding partners of TDP-43, were affected in the presence of the A315T mutation. Exon array analysis on brain and spinal cord tissue from TDP-43 transgenic mice showed that global aberrant splicing leads to changes in the ratio of isoforms of genes such as Zmynd11 and Camk2a. We also identified that this abnormal splicing event occurs presymptomatically and becomes more pronounced as the disease progresses. Further, we noticed certain mRNA targets identified in chapter 3.1 to be potentially irregularly spliced in mouse CNS in the presence of a mutant TDP-43.Finally, by utilising brain tissue samples from ALS patients and controls (obtained from the Queensland Brain bank) we confirmed the aberrant splicing event for human ZMYND11 is similar to observations made for the mouse Zmynd11 gene (chapter 3.4). Even though validation has only been carried out for one gene (ZMYND11) in the human ALS brain tissue samples, we nonetheless show that the data can be translated from mouse models through to humans. This work showed, that validation of further abnormal splicing events discovered in the mouse tissue should benefit from additional studies in human samples.In summary, from the work carried out in this thesis it appears that TDP-43 acts as a master regulator co-ordinating multiple cellular mechanisms in the mammalian central nervous system. Characterisation of mRNA targets of TDP-43 helped us identify synaptic transmission as one of the mechanisms regulated by the protein. From our transgenic mouse study, it is clear that TDP-43 binds to several mRNA targets and regulates RNA processing, which is affected in the presence of mutant TDP-43. Our results also suggest that abnormal pre-mRNA splicing precedes symptoms of ALS in TDP-43 mice. Any malfunction of TDP-43 will affect the regulation of these many targets thus affecting multiple cellular processes that might contribute to the disease aetiology. We hypothesise that ALS is an RNA metabolism disorder and that targeting RNA processing pathways for drug development and therapy might prove valuable for treating patients with ALS.