Characterisation of the zinc transporter ZnT10 and its role in cellular homeostasis in healthy and Alzheimer's disease brain

Abstract

Zinc is essential to the structure and function of numerous proteins and enzymes so requires tight homeostatic control at both the systemic and cellular level. Two families of zinc transporters – ZIP (SLC39) and ZnT (SLC30) – contribute to zinc homeostasis. There are at least 10 members of the human ZnT family, and the expression profile and regulation of each varies depending on tissue type. Little is known about the role and expression pattern of ZnT10; however in silico data predict restricted expression to foetal tissue. In this thesis I show a differential expression profile for ZnT10 in adult human tissue by RT-qPCR and detect highest levels of expression in small intestine, liver and brain tissues. I present data revealing the functional activity of ZnT10 to be in the efflux direction. Using a plasmid construct to express ZnT10 with an N-terminal FLAG-epitope tag, subcellular localisation in a neuroblastoma cell line (SH-SY5Y) is shown to be at the Golgi apparatus under standard conditions of culture, with trafficking to the plasma membrane observed at higher extracellular zinc concentrations of 100 μM. I demonstrate using RT-qPCR down-regulation of ZnT10 mRNA levels in cultured intestinal and neuroblastoma cell lines in response to extracellular zinc, a response which is mirrored at the protein level. Furthermore, I demonstrate reduced transcription from the putative ZnT10 promoter at an elevated extracellular zinc concentration. Reduction in ZnT10 mRNA expression in response to increased extracellular cobalt and conversely, an up-regulation of ZnT10 mRNA levels in response to increased extracellular nickel has also been observed by RT-qPCR in both SH-SY5Y and Caco-2 cells. Neither of these responses were reflected at the level of transcription using the ZnT10 promoter construct. In transiently transfected SH-SY5Y cells Western blotting reveals a reduction in p3xFLAG-ZnT10 protein levels in response to extracellular zinc; however both extracellular cobalt and nickel caused an increase in the level of p3xFLAG-ZnT10 protein measured. Trafficking was observed with both cobalt and nickel treatments. Movement from the Golgi apparatus to a more diffuse localisation pattern was observed with cobalt treatment, whereas nickel elicited a response similar to zinc, with trafficking toward the plasma membrane. Extracellular copper did not induce a change at any level of investigation. These features of ZnT10 localisation, regulation and function, together with the discovery that ZnT10 is expressed at high levels in brain tissue, indicate that ZnT10 has a role in regulating zinc homeostasis in the brain. Further investigation highlighted a down-regulation of ZnT10 in human Alzheimer’s disease brain tissue and in the APP/PS1 transgenic mouse brain. Zinc was also shown to influence splicing events of β-secretase enzyme, known to be involved in Alzheimer’s disease pathology. Zinc promoted an increase in the most active isoform at the mRNA level and thus the measured dysregulation of ZnT10 and therefore potential changes in levels of zinc may have relevance to the development of neurodegenerative disease.