title: Neuroplasticity of the dendritic spine : early dysfunction in amyotrophic lateral sclerosis
Creators: name: Handley, EE
Publisher: name: University of Tasmania
Year: 2019

description: Neurodegenerative diseases are heterogeneous disorders that share common features of progression, whereby vulnerable cortical regions and neuronal populations are susceptible to disease insults during a prolonged preclinical period. At symptom onset, it is theorised that potential compensatory mechanisms mediating these disease insults fail, and overt symptoms then arise. Synaptic dysfunction is one such pathological pathway that has been repeatedly identified in a range of neurodegenerative diseases, yet the initiating mechanism resulting in the dysregulation of synaptic connections has not been identified. Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease, and features the select vulnerability of the motor system and degeneration of motor neurons, for which we have no effective cures or treatments. Critical insight as to which processes may underlie disease onset and progression have come from studies identifying early changes to the excitability of the motor cortex of ALS patients, and increasingly the dysfunction of the synapse has been linked to these network changes. Thus there is an increased interest in the potential for early dysregulation of the synaptic compartment to initiate pathological disease pathways during preclinical periods. The primary component of characteristic disease aggregates in ALS patient tissue is the DNA/RNA-binding protein TDP-43; in disease states, the protein mislocalises to the nucleus and is sequestered into toxic inclusions within the cytoplasm. Though previous research has focused on the role of TDP-43 within the nuclear and cytoplasmic compartments, increasing evidence indicates the protein has normal roles more distally- particularly at the dendritic spine. The dendritic spine is the primary postsynaptic compartment of glutamatergic neurons and is essential for the regulation of neuroplasticity. Therefore, the aim of this thesis was to investigate how and when misprocessing of TDP-43 may impact the postsynaptic compartment at the dendritic spine, and how this may then impact neuroplasticity. Further, the fundamental features of the motor cortex were examined in order to better understand what may render the region vulnerable to dysfunction mediated by changes at the dendritic spine. The current thesis investigated the potential for misprocessed TDP-43 to mediate dendritic spine dysfunction by first establishing a timeline of synaptic alterations in the motor cortex of the TDP- 43\(^{A315T}\) mouse model of ALS. The aim of this first study was to investigate whether the synapse is dysfunctional over a disease time course, in order to identify the earliest occurring disease events mediated by TDP-43 using immunohistochemical techniques. Subsequently, neuroplasticity of the dendritic spine was investigated within the motor cortex under normal conditions using cranial window surgeries in conjunction with 2-photon laser scanning microscopy (2PLSM), in order to identify features that may potentially underlie the vulnerability of the region to disease-linked pathology in real time. An additional aim of this second study was to investigate the influence of the sex hormone oestrogen and age on dendritic spine populations within discrete cortical regions, to better understand factors that may influence the incidence of disease within populations. The final study investigated real-time changes at the dendritic spine in the presence of the TDP-43\(^{A315T}\) mutation, within male and female mice having undergone cranial window surgeries and 2PLSM. This thesis determined that synaptic dysfunction is a presymptomatic disease event in the TDP-43\(^{A315T}\) mouse, occurring specifically in the motor cortex. Dendritic spine density was significantly reduced prior to overt symptoms and cell loss, occuring concurrently with a hypoexcitable phenotype. These changes were targeted within layer V pyramidal neurons of the motor cortex at P60, prior to dendritic spine density changes being evident by P90 within both the motor and somatosensory cortices. These findings highlight the select vulnerability of the motor cortex to TDP-43-mediated dysfunction at the dendritic spine, and thus the features that render this region susceptible to disease were next investigated. This was explored investigating real-time changes in dendritic spines within the motor and somatosensory cortices of Thy1-YFP mice, to establish a neuroplasticity ‘signature’ for each region. The dendritic spines of the motor cortex were found to be highly dynamic from P60 through to P90 in both male mice and females experiencing baseline levels of oestrogen, whilst during cycling peaks of oestrogen the dendritic spines of the motor cortex were observed to be stable. Conversely, the somatosensory cortex displayed early stability within males, prior to higher dynamics at adulthood as seen in females. These findings revealed that spine dynamics were specific to brain region, and that in the motor cortex dependent upon cycling oestrogen. Within the motor cortex of TDP-43\(^{A315T}\) mice at P60, males displayed significantly reduced dynamics, whilst high oestrogen females were associated with increased dendritic spine dynamics. Collectively, these findings indicate the sustained dynamics of the motor cortex in normal conditions may prime the region to fail in the presence of activitydependent pathology at the dendritic spine. The potential for cycling oestrogen to be neuroprotective at the dendritic spine under these conditions is evident, and highlights the need for further studies to explore this as a potential therapeutic pathway. The findings from this thesis provide strong evidence in support of the crucial role neuroplastic mechanisms at the dendritic spine have in response to pathology. Here, evidence is provided for the early dysfunction of the synapse in an ALS disease model being a key disease event within the presymptomatic motor cortex, a region that may be primed to fail fundamentally as a result of sustained activity demands at the dendritic spine. Further, the thesis supports the notion of oestrogen as being neuroprotective at the synapse, and indicates the transient peaks of the hormone maintain compensatory dynamics in the presence of misprocessed TDP-43. These results highlight the targeted nature of vulnerability to neurodegenerative diseases, and highlight the need to target early dysregulation of neuroplasticity at the synapse to protect select cortical networks prior to neurodegeneration. Importantly, the identification of cycling oestrogen as being protective at the dendritic spine may be critical for future efforts aimed at developing new therapeutic targets in ALS and other neurodegenerative diseases; for this application to be viable, it is essential to investigate further the role for disease proteins at the dendritic spine, as well as the mechanisms through which oestrogen exerts neuroprotective effects.