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

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

An efficient and cost-effective method of generating postnatal (P2–5) mouse primary hippocampal neuronal cultures

Background: Primary culture of postnatal central neurons is a widely used methodology for applications such as the investigation of neuronal development, protein trafficking/distribution and cellular signalling. However, successful production and maintenance of such cultures, particularly from postnatal animals, can be challenging. In attempting to surmount these difficulties, several disparate culturing methodologies have been developed. Such methodologies are centred on the identification and optimisation of critical steps and, as such, the protocols and reagents utilised can differ quite markedly from protocol to protocol, often with the suggestion that the use of a (usually expensive) proprietary reagent(s), lengthy substrate preparation and/or cell isolation techniques is/are necessary for successful culture preparation. New method: Herein, we present a simple and inexpensive protocol for the preparation of primary hippocampal neurons from postnatal (2–5 day old) mice, which remain viable for experimental use for over one month. Results: Neurons cultured using this method follow well established developmental norms and display typical responses to standard physiological stimuli such as depolarisation and certain pharmacological agents. Comparison with existing methods/conclusion: By using a novel trituration technique, simplified methodology and non-proprietary reagents, we have developed a reliable protocol that enables the cost effective and efficient production of high quality postnatal mouse hippocampal cultures. This method, if required, can also be utilised to prepare neurons both from other regions of the brain as well as from other species such as rat

Routine plain film of abdomen in asymptomatic patients with video capsule retention in small bowel-time to change practice?

Aim: Small bowel capsule retention is rare, with a rate of approximately 2%, defined as visible retention on plain film of abdomen (PFA) after 14 days. Currently, PFA is performed if the capsule is not seen to reach the large bowel during recording. Alternatively, for upper gastrointestinal (UGI) capsule studies, the risk of retention is determined if the capsule fails to reach the small bowel during recording. Given the similar physical specifications of the capsules (Medtronic) used, we considered whether 14-day PFA is no longer required for small bowel capsules not observed in the large bowel.Methods: The use of patency capsules in our lab allows careful selection of small bowel capsule studies to minimize the risk of retention. All PFAs performed over a five-year period were reviewed to determine if careful selection and use of patency negate the need for capsule retention PFA screening.Results: In total, 688 small-bowel capsules were performed during the study period, and 3.6% had prior patency capsules. Thirty-one PFAs with a query of capsule retention were performed during the study period on 28 patients. This included 15 females, and the median age was 53.5 years. None of the films demonstrated capsule retention.Conclusion: Our data suggest that 14-day PFA may no longer be required for small bowel capsules not seen to reach the large bowel. Advice regarding symptoms of capsule retention and precaution with magnetic resonance imaging, similar to current UGI capsule advice, may suffice. This may reduce the burden on radiology imaging slots and, in particular, eliminate unnecessary radiation exposure and repeat hospital attendance for patients