Mechanism of action and functional role of the two-pore channels (TPCs) in key vertebrate physiological processes

Thesis

Two-pore channels (TPCs) represent a strong candidate for being pivotal components of the endolysosomal cation channels regulated by nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca²⁺-releasing second messenger. A substantial body of evidence has implicated TPCs functional roles in key physiological processes, such as autophagy, osteoclast formation, neuronal differentiation, etc. Given the importance of Ca²⁺ signals in regulating skeletal muscle development and differentiation, and the rising prominence of NAADP/TPC signalling in regulating different aspects of physiological events, the first part of my research project sought to determine whether this lately-established Ca²⁺ mobilization also plays a role in myogenesis. By using morpholino-based knockdown of TPC2, together with bioluminescence-based Ca²⁺ imaging, a significant disruption of the characteristic Ca²⁺ signalling profiles generated by slow muscle cells of intact zebrafish embryos was noted. In addition, impairment in the gross and fine structure of the trunk of zebrafish embryos was apparent in TPC2 morphants. Thus suggesting involvement of TPC2 in regulating slow muscle cell development. On the other hand, whole-mount in situ hybridisation of intact zebrafish embryos revealed an expression of Tpcn3 mRNA, implicating TPC3 a role in zebrafish embryogenesis.

Previous study showed that TPC2 was transiently upregulated during the early stages of C3H10T1/2 adipocyte differentiation in vitro, the second part of my project aimed to investigate whether TPC2 plays a functional role in such process, also to identify the upstream mediators involved in regulating Tpcn2 expression and potential downstream effectors mediated by TPC2. My findings demonstrated impairment of adipogenesis in TPC2 knockdown cells and in cells depleted of lysosomal stores, indicating an involvement of TPC2 and lysosomal stores in such process. Besides, PKA and Epac were found to be required for the observed Tpcn2 upregulation, probably via a mechanism involving CREB activity, based on the findings that CREB bound to CRE element of Tpcn2 promoter. Preliminary results further suggested that calpain might be a potential effector of TPC2 in regulating C3H10T1/2 adipogenesis.

In view of the accumulating evidence showing the participation of NAADP in the nervous system, the third part of my work presented evidence of expression of Tpcn1 mRNA in several regions of the brain, implicating potential role(s) for TPC1 in those areas.

Authors: Chan, L

• DOI: 10.5287/ora-pvkjxbo6j
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford