Investigations into the distribution and function of nucleobindin 1

Abstract

The nucleobindin proteins (NUCB1 and NUCB2) are both characterised by DNA- and calcium-binding motifs. The functionality of these motifs has been demonstrated, but has not yet led to the identification of a role for either protein. The vast majority of literature on NUCB2 has somewhat overlooked these motifs by pursuing the idea that the protein is cleaved to yield a secretory N-terminal peptide fragment. These studies have implicated NUCB2 in a range of physiological processes and diseases, but no mechanism of action has yet been suggested and, furthermore, the endogenous existence of the peptide fragment remains controversial. Meanwhile, investigations into NUCB1 have proposed a number of interaction partners, but have also been unable to define a precise action for the protein.

In this thesis we set out to investigate the distribution and function of NUCB1. Exploring the expression of the protein in the nervous and endocrine systems, we found that NUCB1 exists in a wide array of tissues, but appears to be specifically expressed in secretory cells. This pattern was consistent across the nervous system, where the protein was found in all neuronal populations examined, but absent from glial cells. Similarly, within the endocrine tissues NUCB1 appeared to be restricted to the hormone producing cells, for example the islets of Langerhans, but was devoid from other cell types, such as the exocrine acinar cells. In the pancreatic beta cells and certain populations in the hypothalamus, NUCB1 and NUCB2 were found to be co-expressed, indicating that, despite the structural similarity between these two proteins, they may perform diverse functions.

In order to explore the function of NUCB1 we first developed an organotypic slice culture model of the hypothalamic tuberoinfundibular dopamine (TIDA) network. The electrophysiological properties of these cells have been well characterised, providing a good platform from which to investigate the impact of modulating NUCB1 expression. Furthermore, like the beta cells, the TIDA neurons express both NUCB1 and NUCB2, making both systems useful for studying possible complementary or competing activities of these related proteins. Comparison of the TIDA neurons in organotypic cultures and acute slices revealed that the electrophysiological and morphological properties of these cells are largely unaffected by the culture process, suggesting that the organotypic slices provide a reliable model for functional investigations.

Employing both the organotypic TIDA and isolated pancreatic islet models, we explored the role of NUCB1 in secretory cells. Although altering the expression of NUCB1 was not found to impact cell transcription or calcium handling in the islets, we discovered that knockdown of NUCB1 in the TIDA neurons reduced the frequency of excitatory and inhibitory post synaptic currents. This change could not be attributed to a difference in cell morphology, indicating that NUCB1 may be involved in cell signal transmission.

The work presented in this thesis demonstrates that NUCB1 is broadly expressed in secretory cells and suggests that it may contribute to the signalling responses of these cells. Further investigations are required to elucidate what this contribution may be and whether these findings are consistent with other cells expressing NUCB1.