Inhibiting the Notch signalling pathway with either the γ-‐secretase inhibitor DAPT or following siRNA knockdown of RBP-‐Jκ was found to result in an almost 8-‐fold increase in the expression of the immature neuronal marker Tuj, with positive cells demonstrating a typical neuronal phenotype. Unsurprisingly, this increase mirrors the findings in ENS mouse models in which Notch is inhibited via either a PTCH1 or POFUT1 knockout confirming that these studies were reflecting the consequences of Notch inhibition rather than some other unidentified effect of the knockouts (Okamura, et al 2008, Ngan, et al 2011).
Importantly, Notch inhibition also promoted the expression of the more mature neuronal marker nNOS. Unsurprisingly this lagged behind the expression of the immature marker Tuj, with nNOS showing a significant increase after 96h compared to 24h. The overall expression of ChAT was not significantly higher in Notch inhibited cells than in controls after the full 192h incubation period, even though there had been a significant increase in the proportion of cells expressing ChAT (4-‐fold increase between 96h and 192h).
The development of nitrergic and cholinergic neurons is known to differ. During embryonic development ENS cholinergic neurons differentiate and exit the cell cycle earlier than their nitrergic counterparts (Pham, et al 1991). Although, there is a paucity of evidence detailing the timing of nitrergic and cholinergic differentiation in-‐vitro, the literature suggests that nitrergic differentiation may occur earlier. Papers report increases in the expression of nNOS after a period of 24-‐48h (Anitha, et al. 2010), whereas increases in ChAT are reported after longer time periods, e.g. 18 days (Nilbratt, et al. 2010). However, these observations must be treated with caution as there are no studies providing a direct comparison between cholinergic and nitrergic differentiation in-‐vitro under controlled conditions.
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It has been shown that neuronal subtypes respond differently to inflammatory insults within the gut (Winston, et al. 2013)and it is equally plausible that these neuronal subtypes respond differently to changes Notch signalling. Preliminary evidence from Sander et al provides some support for this premise, in finding differential expression of the Notch 1 receptor between enteric cholinergic and nitrergic neurons. However their report was limited to in-‐situ hybridisation results of one single Notch receptor and did not attempt to correlate these findings with function.
It is not clear from these results whether the Notch is just responsible for promoting the initial switch to an immature neuronal lineage, or whether prolonged inhibition is required to promote mature neuronal differentiation. There is not enough evidence provided in this chapter to draw any definitive conclusions as to whether these results are due to differences in the timing of differentiation or the response to changes in Notch signalling. However, it does warrant further study because if it is possible to promote the differentiation of inhibitory neurons in progenitors prior this may improve the functional response of any future cell-‐based transplantation therapies.
4.3.4. Notch signalling is disrupted by dissociation of neurospheres
In this chapter we have used standard immunofluorescence techniques to detect the cleaved form of NICD, to determine the activation state of the Notch signalling pathway. We found that the proportion of cells in which NICD could be detected fell quickly after dissociation, a process that was accelerated by Notch inhibition. This decrease in the expression of cleaved NICD following dissociation is not unexpected. Notch signalling is classically activated by transmembrane ligands on adjacent cells, hence disruption of cell-‐ cell interactions will interrupt canonical Notch activation. Following dissociation, the proportion of cells expressing NICD under uninhibited conditions gradually increases again,
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as the cells become more confluent. Although, the expression of NICD does fall following either cell dissociation or Notch inhibition it is not immediate. Other groups have used the expression of NICD as a proxy for Notch activation (Hansson, et al. 2006, Del Monte, et al. 2007), however it still has its drawbacks, the detection of cleaved NICD may lag behind the true activation state, for example, although the antibodies only detect the cleaved form of NICD, it is unclear how long NICD may remain detectable following cleavage before it is broken down and recycled. Furthermore, the Notch signalling pathway can be activated in a non-‐canonical, NICD independent, manner (Martinez Arias, et al. 2002), which this technique would not detect. In order to more precisely determine the changes in Notch activation ‘live-‐reporting’ techniques such as using a luciferase-‐base reporter to assess levels of activated RBP-‐Jκ would be required (Ilagan, et al. 2011).
The presence of cleaved NICD was also used to determine activation firstly within the microenvironment of the neurosphere, where its distribution is predominantly in cells at the periphery, suggesting that Notch signalling is most active in this region, which itself is consistent with the previous findings regarding the expression of Notch receptors and ligands (4.2.1). Secondly, it is also seen within the ENS in-‐vivo, which is more surprising. The initial hypothesis was that Notch was inactive in-‐vivo, and following an ‘injury’, such as isolating neural progenitors, it became active thus explaining the increase in proliferation and maintenance of a stem cell like state. If cleaved NICD is present in-‐vivo this may suggest that Notch is necessary but not sufficient for progenitor cell self-‐renewal in-‐vivo. More importantly, it suggests that the environment in-‐vivo exerts a dominating inhibitory action on ENS progenitor cell self-‐renewal, irrespective of the activity of the Notch
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