Defining the function of Amyloid Precursor Protein dimerisation in neuritogenesis
Document TypePhD thesis
Access StatusOpen Access
© 2017 Dr. Luan Luu
Amyloid Precursor Protein (APP) is known to be primarily involved with Alzheimer’s disease; however APP is also involved with neurogenesis, synaptic plasticity and neuroprotection. Many factors can bind to APP to affect its function and processing. APP can also bind to other membrane bound APP, known as APP dimerisation. We hypothesise that dimerisation of APP can affect the biological actions of APP. This study aims to determine the effect APP dimerisation has on neurite outgrowth and elucidate its mechanism of action. We found that APP dimerisation can reduce neurite outgrowth by modulating extracellular and intracellular signals regulating neurite outgrowth. We determined the effect APP dimerisation has on APP neurite outgrowth by using APP dimerisation mutants, G33I and L17C (these cause decreased and increased APP dimerisation, respectively), transfected into SH-SY5Y cells. To determine if APP dimerisation utilises extracellular signalling to modulate neurite outgrowth, condition media treatment of APP dimerisation mutants was used to determine if a secreted factor was responsible for modulating neurite outgrowth. Intracellular mechanisms including APP localisation, RhoA activity and miRNA expression were investigated. The localisation of APP can affect its function therefore; immunofluorescent imaging was used to determine its localisation. RhoA GTPase is known to negatively regulate neurite outgrowth, therefore RhoA activity was determined using an ELISA based assay that was specific for activated RhoA. The Ion Torrent Next Generation Sequencing system was also used to determine the differential expression of miRNA in the APP dimerisation mutants and these were confirmed by qRT-PCR. APP-L17C mutants inhibit neurite outgrowth by inhibiting the secretion of a neurite outgrowth promoting factor as APPwt condition media treatment rescues neurite outgrowth. APP dimerisation also caused perinuclear APP aggregates which colocalised within the endoplasmic reticulum. RhoA activity is increased in the APP-L17C mutant, and treatment with condition media decreases RhoA activity which correlated with neurite outgrowth. APP dimerisation also affects several miRNA expressions. The miR-125a, miR-135b, miR-34a species, which have a known role to play in the regulation of neuritogenesis, were down regulated and transduction of an miR-34a restored neurite outgrowth back to wild type levels. In conclusion, APP dimerisation reduces neurite outgrowth and can mediate its effects by inhibiting a secreted factor, modulating APP localisation, increasing RhoA activity and modulating the expression of certain miRNAs involved in neurite outgrowth.
KeywordsAmyloid Precursor Protein; dimerisation; neurite outgrowth
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