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- Title
Expression and characterization of the human amylin receptor AMY3 in the mammalian cell-line RK13 (rabbit kidney cells) as a cellular model for Amyloid beta neurotoxicity in Alzheimer's Disease.
- Authors
Isaac, Andre; Wen Fu; Jhamandas, Jack H.
- Abstract
The neurotoxic effects of amyloid β protein (Aβ) have been well documented as a key pathological process in Alzheimer's Disease (AD) (Hardy et al., Science, 2002). Although no receptor has been explicitly identified for Aβ, recent studies from our laboratory have demonstrated that Aβ may express its toxicity through the human amylin receptor, which is composed of the human calcitonin receptor (CTR) and a receptor-associated membrane protein (RAMP 3) (Jhamandas et al., Neuroscience, 2004). There is also evidence to suggest that activation of human amylin receptor complex elicits signal transduction effects via the Gas-cAMP pathway (Morfis et al., Endocrinology, 2008). However, it is difficult to isolate the pharmacological and molecular aspects of the amylin receptor activation in neurons due to the complexity of such cell systems. We introduced the gene fragments necessary to express the human amylin receptor in a mammalian cell-line (rabbit kidney cells - RK13) in order to produce a simple cellular model, which is a necessary step to unequivocally establish the human amylin receptor as a target for Aβ. Both CTR and RAMP3 cDNA were inserted into a modified pBud-CE4 vector with GFP tag and transfected into RK13 cells. Transfection was confirmed by fluorescent tagging with GFP expression. A stable cell-line was developed by using zeocin antibiotic selection. Transfected cells were exposed to human amylin, and the resultant cAMP signal was detected via immunohistochemistry and compared to that for non-transfected, wild-type cells. Electrophysiological patch clamp recordings also suggested a link between the transfected amylin receptor and potassium ion conductances when the cells were exposed to human amylin. These data indicate that the transfected RK13 model can be used to express the human amylin receptor, and this system will be essential for future research into the cellular effects of β, and its involvement in AD pathogenesis.
- Subjects
ALZHEIMER'S disease; AMYLOID beta-protein; AMYLIN; NEUROTOXIC agents; NEUROTOXICOLOGY
- Publication
UBC Medical Journal, 2011, Vol 2, Issue 2, p38
- ISSN
1920-7425
- Publication type
Abstract