Investigating neuronal calcium homeostasis in murine models of Alzheimer’s disease

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Kaar, Aidan
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University College Cork
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This thesis is primarily concerned with the pathological remodelling of calcium homeostasis and signalling in murine Alzheimer’s disease (AD) models, namely the 3xTg-AD (APPSWE/PS1M146V/TauP301L) mouse and TgF344-AD (APPSWE/PS1∆E9) rat models. The majority of data contained herein was amassed from calcium imaging of primary neuronal hippocampal cultures. The development of a new protocol for the primary culture of mouse hippocampal neurons also represents a major research output of this thesis (Kaar et al., 2017). In addition to simplifying and optimising each facet of the culturing process, we identified the mechanical dissociation of hippocampal tissue (trituration) as the critical factor in determining the success or otherwise of culture preparations. Furthermore, we demonstrated that successful primary mouse hippocampal cultures could be prepared without the need for specialised and expensive proprietary supplements which many researchers in the field propose as being essential for this purpose. As such, this method fills a niche in the current published literature where efficient, cost-effective and succinct methods for postnatal mouse cultures are lacking. Calcium imaging experiments were predominately conducted using cultures produced from three to 6-day-old mice and rats. In using animals at such a young age it is thought that any observed calcium dysregulation may reflect early pathological or perhaps even initiating AD events. In line with the “Calcium hypothesis of Alzheimer’s disease” (Berridge, 2010), this research points to cellular Ca2+ remodelling in terms of both basal Ca2+ levels and Ca2+ signalling as an early pathogenic event in AD. In particular, we have studied how the main neuronal calcium store, the endoplasmic reticulum (ER), functions in both maintaining calcium homeostasis and mediating intracellular signalling processes, and how these functions might be disrupted in AD. Using calcium imaging, group 1 metabotropic glutamate receptor (I-mGluR)-mediated somatic responses were measured under basal conditions and also under conditions where the ER was ‘preloaded’ with calcium (using a depolarising stimulus (15mM K+) which, we propose, mimics a ‘learning event’ in the hippocampus). In non-transgenic neurons from both murine models, I-mGluR activation combined with the loading stimulus-evoked enhanced Ca2+ signals relative to I-mGluR activation alone. In contrast, enhanced calcium responses were not observed in 3xTg-AD mouse hippocampal neurons, suggesting a loss of this signalling function. Notably however, under basal conditions, we did observe significantly larger responses to I-mGluR activation as well as attenuated SOCE in 3xTg-AD neurons suggesting a pathological increase in resting ER calcium levels in these neurons relative to controls. Furthermore, in TgF344-AD neurons, there were also subtle differences in I-mGluR response parameters when compared with non-Tg controls. The fact that such stark alterations in calcium homeostasis and signalling were observed in neurons from 3xTg-AD mice at such a young age (≤6 days), suggests that calcium dysregulation may occur at a much earlier stage in the disease progression than previously thought and appear to greatly precede the appearance of Aβ or tau pathology. The third major theme of this thesis involved identifying the downstream signalling cascade(s) involved in I-mGluR activation. The classical understanding of I-mGluR activation involves phospholipase C β (PLCβ)-mediated production of IP3 and Ca2+ release from the ER via the IP3 receptors (IP3R). However, we show here that a significant contribution (approximately 10 - 40%) of I-mGluR mediated Ca2+ mobilisation from the ER is in fact mediated by the second messenger, cyclic ADP-ribose (cADPR), which culminates in RyR-mediated Ca2+ release. Furthermore, the relative contribution of both PLCβ/IP3R and cADPR/RyR pathways to overall I-mGluR elicited signals is altered in transgenic neurons of both murine models. Finally, the expression of key calcium toolkit components including relative IP3R and RyR isoform expression, as well as the expression of Bcl-2 and Bcl-XL, which modulate ER Ca2+ release channel function, was determined in hippocampal tissue from 5-, 15- and 21-day-old 3xTg-AD mice. It was found that there was a trend towards decreased expression of Bcl-2 and Bcl-XL and a trend towards increased expression of RyR and IP3R1 with development (P5 vs P15 and P5 vs P21) regardless of transgenic status, however, this was significant solely in 3xTg-AD tissue and only in the case of Bcl-2, Bcl-XL and RyR (2) expression (and generally, solely at the latest stage of development tested, P21). In summary, the work set out in this thesis investigates neuronal Ca2+ homeostasis and I-mGluR/ER signalling as well as relevant protein expression with a particular focus on possible AD-mediated dysregulation of these processes. Lastly, the method of primary hippocampal culture, the sources of experimental units from which this data has been accrued has been extensively optimised.
Primary neuronal culture , Hippocampal neurons , Trituration , 3xTg-AD mouse , TgF344-AD rat , Presenilin , Endoplasmic reticulum , Metabotropic glutamate receptor , mGluR , I-mGluR , cADPR , ADP ribosyl cyclase , glutamate , Bcl-2 , Bcl-XL , IP3R , RyR , Alzheimer's disease , Calcium signalling , Calcium homeostasis , Calcium dysregulation , The calcium hypothesis of AD , Glutamate
Kaar, A. 2019. Investigating neuronal calcium homeostasis in murine models of Alzheimer’s disease. PhD Thesis, University College Cork.
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