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Cellular dysfunction in amyotrophic lateral sclerosis : investigating the role of TDP-43

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Jiang, T 2020 , 'Cellular dysfunction in amyotrophic lateral sclerosis : investigating the role of TDP-43', PhD thesis, University of Tasmania.

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Abstract

The discovery that most pathological protein inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) contain the aggregated transactive response DNA-binding protein 43 kDa (TDP-43) has resulted in significant research investigating the role this protein may play in the progressive neurodegeneration, underlying genetic factors and molecular characteristics of these now so-called TDP-43 proteinopathies.
Despite these research efforts, there is still no cure for ALS and the two available therapeutics extend survival by mere months. ALS is the most common motor neuron disease (MND) and traditionally is characterised by selective degeneration of both upper and lower motor neurons (MNs) and the subsequent systematic destruction of the motor system. This results in a multitude of motor symptoms, including muscle weakness, spasticity, fasciculations, and eventually respiratory dysfunction and failure.
Although recent research has proposed an important role for TDP-43 in the formation and maintenance of the nervous system, the molecular mechanisms underlying how TDP-43 affects pre- and post-synaptic compartments which make up neuronal connections in the motor system are not fully understood.
Autosomal dominant point mutations in the TARPD gene that encodes TDP-43 are linked to both sporadic and familial ALS, indicating that alteration of TDP-43 can cause neurodegeneration directly (Yokoseki et al., 2008). One such point mutation is the A315T mutation. This ALS-linked mutation was one of the first utilised to create a mouse model of TDP-43 pathogenesis. In this model, driving human TDP-43 with the A315T point substitution mutation on the prion promotor causes a selective loss of upper motoneurons and lower motor neurons, as well as loss of function on the rotarod motor function test and muscle atrophy (Wegorzewska, Bell et al. 2009, Guo, Wang et al. 2012, Handley, Pitman et al. 2017).
In the last decade the classification of ALS has transformed from being a pure MN disease to be considered instead a complex neurodegenerative disease, where both excitatory and inhibitory imbalances are likely to contribute to the disease manifestations. This has been characterised by degeneration of inhibitory cortical circuits together with increased excitation of excitatory systems. Cortical hyperexcitability has been demonstrated to develop prior to clinical symptoms, suggesting that it may contribute to subsequent neurodegeneration. Indeed, increased cortical hyperexcitability of both familial and sporadic ALS patients has been investigated, which proposed that altered motor network excitability may be a central pathogenic mechanism in the disease, possibly initiating the final progressive decline of motor neuron function. However, it is not fully understood whether the misprocessing of TDP-43 alters synaptic signalling and could be driving these pathogenic alterations in excitability that lead to neuronal dysfunction and neurodegeneration.
This thesis examines the potential role of TDP-43 in disease by using primary cortical neurons derived from the TDP-43\(^{A315T}\) mouse model of ALS. The intent of this initial study was to determine why cortical neurons are vulnerable in ALS patients and how excitotoxicity contributes to the pathogenesis of ALS. Subsequently, the effects of TDP-43\(^{A315T}\) on the postsynaptic compartment was established through dendrite spine and post synaptic protein analysis in vitro. An additional aim of this study was to determine the effects of TDP-43\(^{A315T}\) at the pre-synapse, by using microfluidic devices. The final study investigated the potential effects of pathological TDP-43 in muscle cells, again using an in vitro approach, but in this instance with the C2C12 myoblast cell line, derived from adult mouse statellite cells, was transfected with small interfering RNA (siRNA) of TDP-43.
To determine the effect of mutant TDP-43 on the development and formation of synapses in excitatory neurons in vitro, we derived primary cortical neuronal cultures from transgenic mouse embryos expressing human TDP-43\(^{A315T}\) under the Prp promoter and yellow fluorescent protein (YFP) in cortical pyramidal/projection neurons under the Thy1 promoter.
In Chapter 1, we describe our examination of the neuronal process structure and dendritic spine density in vitro. Immunocytochemistry using an antibody MAP2 to label dendrites was performed, in conjunction with subsequent cell tracing, in order to assess both dendritic
morphology and spine density.
Chapter 2 details how we determined if axonal development was impaired in the presence of the TDP-43\(^{A315T}\) mutation in vitro. For these analyses, microfluidic chamber cultures, immunocytochemistry, live imaging and real time qPCR techniques were used. The characterisation of isolated axons in vitro allowed the evaluation of not only morphological alterations, but also insight into functional changes.
The final Chapter describes the work to determine the effect of knockdown of TDP-43 on the differentiation of C2C12 cells.
Collectively, the study findings established that ALS-linked TDP-43\(^{A315T}\) has a significant pathological influence on synapse development in pyramidal cortical neurons, without influencing muscle cell differentiation. This determination supports the hypothesis ‘TDP-43 is involved in synapse development and formation by acting at the formation of the ionotropic glutamate receptors in cortical neurons.

Item Type: Thesis - PhD
Authors/Creators:Jiang, T
Keywords: ALS, TDP-43, Axon, Dendrite, GluR
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Copyright 2019 the author

Additional Information:

Chapter 2 is related to an article published as: Jiang, T., Handley, E. E., Brizuela, M., Dawkins, E., Lewis, K. E., Clark, R. M., Dickson, T. C., Blizzard, C. A. 2019. Amyotrophic lateral sclerosis mutant TDP-43 may cause synaptic dysfunction through altered dendritic spine function, Disease models & mechanisms, 12(5), 1-11. It is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License, (https://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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