Abstract:
Huntington’s Disease (HD) is an inherited autosomal-dominant, incurable neurological disorder. The three primary symptoms of HD are motor function impairment, psychiatric disturbances and cognitive decline. Recently, a widespread increase in urea has been observed in post-mortem brain tissue from HD patients. Furthermore, in a transgenic sheep model of early HD, this increase in urea is restricted to the striatum, the primary site of HD pathology. RNA sequencing of the same sheep reveals an increase in SLC14A1, a urea transporter and RHCG, an ammonia transporter, in neurological tissue. In addition AQP9, a water transporter which can facilitate urea movement has recently been identified as a biomarker for HD. Therefore, the aim of this project is to advance the understanding of the elevation of urea in HD patients by examining urea and ammonia transporters specifically, AQP9, SLC14A1 and RHCG. Three integral concepts were investigated to achieve this aim; to generate plasmid vector to express human AQP9, SLC14A1 and RHCG proteins, to work towards the development of a cell culture model which can facilitate understanding factors that affect urea production and identifying if the selected transporter proteins are differentially expressed in human brain tissue. Modified plasmids expressing RHCG were successfully generated, and the transporter proteins expressed and identified using transfection and Western blot analysis. In comparison, expression of SLC14A1 and AQP9 proteins from modified plasmids could not be confirmed. In cell cultures, ammonia, media composition and incubation time variables were investigated to identify if they impact urea production. In a hepatocyte cell line, HEP3B, low levels of ammonia (6-12mM) in a high nutrient rich media (GlutaMAX), in short, incubation times (18- 24 hours) resulted in detectable urea production. In a large cohort of HD and control postmortem brain tissue, SLC14A1 protein expression level was not significantly altered, nor was there a relationship between SLC14A1 protein and urea. Interestingly, in a preliminary study of RHCG, protein expression in a small human cohort, the predicted immunopositive band was increased in patients compared to controls. Observations from this project indicate that RHCG provides a novel pathway for understanding HD pathogenesis.