Ko, Suh Youn (2017) Effect of Netrin-1 on the mature enteric nervous system and colorectal cancer. PhD thesis, Victoria University.

Abstract

The enteric nervous system (ENS) plays a pivotal role in regulating gastrointestinal functions including intestinal wall movement, water/electrolyte absorption and secretion. Clinical studies have alerted that enteric neuropathy is induced by colorectal cancer (CRC), and this deficit may be a primary reason for CRC patients experiencing clinical symptoms such as constipation and diarrhoea. Manifested clinical symptoms are likely to be seen in CRC patients undergoing chemotherapy, as chemotherapeutic agents are known to be associated with neurotoxicity as a side-effect. Despite this, no efficient treatment for enteric neuropathy is currently available. This heightens the importance of developing neuroprotective treatment. This thesis hypothesised that Netrin-1 could be an ideal candidate as a neuroprotective agent, as it is a well-known axonal guidance cue in the developing central nervous system, and its expression in the adult nervous system is shown to be involved in nerve regeneration after injury. It is recently found that Netrin-1 plays a guidance role in the developing ENS for establishing vagal afferent innervations to the gut. However, the role of Netrin-1 in the mature ENS is yet to be elucidated. In addition to the nervous system, Netrin-1 is found to play a regulatory role in tumourigenesis. This therefore makes the use of Netrin-1 complicated, and thus far it is unclear whether Netrin-1 can be used therapeutically. This thesis aims to investigate the effect of Netrin-1 on adult enteric neurons and colorectal cancer cells at a cellular level, and uncover the impact of Netrin-1 treatment on the ENS under cancer condition in an in vivo model. The results of in vitro studies, shown in this thesis, demonstrate that Netrin-1 exerts neurotrophic guidance for post-natal enteric neuronal precursor cells by an increase in migration and neurite outgrowth, whilst no apparent effect of Netrin-1 was noted on differentiated neurons. Furthermore, Netrin-1 treatment inhibited caspase-3 activation, which was induced by CRC secretion, in precursor cells. In addition, the effect of Netrin-1 on murine CRC cells indicated that Netrin-1 plays a role in tumourigenesis. Netrin-1 activated pFAK/pMEK/pERK signalling pathway in colorectal cancer cells via UNC5H2 and Neogenin receptors, resulting in increased proliferation, adhesion and migration. In contrast to these effects of Netrin-1 shown in in vitro models, the in vivo study of this thesis demonstrated that a high concentration of Netrin-1 treatment in CRC mice inhibited tumour growth. Furthermore, Netrin-1 treatment did not affect any obvious changes in the ENS component of the CRC mice. A potential reason why the ENS was unaffected by Netrin-1 treatment could be due to the following new findings shown in this thesis. Namely, that Netrin-1 is intrinsically expressed in the mature myenteric plexus of the colons in mice. Specifically, Netrin-1 and DCC receptors are present and co-localised in almost all ganglia and processes of the mature myenteric plexus of the colon in healthy and CRC conditions. Furthermore, Netrin-1 expression was found to correlate with ChAT and nNOS phenoptypes in healthy and CRC conditions. Differential expression of Netrin-1 and changes in the distribution of Netrin-1 expression in relation to ChAT were found to be evident in CRC condition. Collectively, this thesis provides groundwork for a potential therapeutic use of Netrin-1 as a neurotrophic factor for the adult enteric neurogenesis, and exhibited a potential safe use of Netrin-1 treatment for the ENS in the context of cancer.

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