Investigation of endocytic trafficking in the zebrafish neuroepithelium

Abstract

OCRL1 is an inositol polyphosphate-5-phosphatase which regulates levels of phosphatidyl-inositol-4,5-bisphosphate, a membrane lipid important for numerous cellular processes. Mutations in OCRL1 are responsible for Lowe syndrome, an X-linked autosomal recessive disorder which specifically affects the eyes, kidneys and CNS. Despite increased understanding of OCRL1’s cellular role, the pathophysiological mechanisms underlying Lowe syndrome are not well understood. Zebrafish are a good organism in which to study OCRL1 function in vivo. In order to investigate whether loss of OCRL1 impairs endocytic trafficking events important for neuronal development and adult CNS homeostasis, the role of zebrafish OCRL1 in endocytosis, early endosome function and lysosomal function was investigated. This thesis shows that OCRL1 plays a key role in regulating endocytic trafficking within neuroepithelial cells of the developing CNS. OCRL1 is seen to localise to early endosomes and overlap with the endocytic cargo megalin within the neuroepithelium. Consequently, early endosomes are enlarged in OCRL1 mutant zebrafish and trafficking of megalin is impaired, with reduced levels at the apical membrane. Establishment of an assay to test neuroepithelial endocytic uptake revealed that endocytosis is unaffected by loss of OCRL1 function. These results demonstrate that OCRL1 is important for proper functioning of the early endosome within the neuroepithelium and suggest that dysregulated endocytic trafficking may contribute to Lowe syndrome CNS pathology. The role of OCRL1 in CNS lysosomal function was also investigated based on recent reports implicating OCRL1 in regulating lysosomal levels of PtdIns(4,5)P2 during autophagy. Minimal amounts of OCRL1 localised to neuroepithelial lysosomes at both steady state and following induction of autophagy. Furthermore, absence of OCRL1 in OCRL1 mutant zebrafish did not impair lysosomal homeostasis or autophagosome turnover suggesting the function of OCRL1 at the lysosome may not be relevant to Lowe syndrome pathology within the CNS.

Date of Award	31 Dec 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Martin Lowe (Supervisor) & Adam Hurlstone (Supervisor)