Pathological Mechanisms Underpinning Amelogenesis Imperfecta in Mice carrying an Amelogenin Mutation

Mice carrying a Y64H amelogenin mutation phenotypically mimic human amelogenesis imperfecta. Affected ameloblasts are characterised by the presence of abnormal cytoplasmic vesicles of retained amelogenin. Protein-protein binding studies using recombinant wild type and Y64H amelogenin revealed that the mutation increased amelogenin-amelogenin binding. This may drive intracellular aggregation of Y64H amelogenin, explaining the abnormal retention. Intracellular protein aggregation causes ER stress which triggers the UPR. The UPR attempts to restore proteostasis but as a last resort triggers apoptosis. SEM of affected enamel showed initially secreted enamel is normal; coincident with UPR in pro-survival mode. The final outer enamel is abnormal; indicative of UPR induced ameloblast apoptosis. Q-RT-PCR was used to measure ER stress related gene expression in affected ameloblasts. Expression levels of ER stress genes increased but not significantly (significance was reached in later studies by others in the research consortium). Amelogenin expression was shown to be significantly reduced in affected ameloblasts; reduced protein expression being a known pro-survival tactic employed during ER stress. A steady-state in vitro mineralisation system was used to examine the effect of the Y64H mutation on mineral nucleation by recombinant amelogenins in isolation or in conjunction with recombinant 32 kDa enamelin. Data showed that the Y64H mutation did not affect the nucleating potential suggesting that the pathological mechanism driving AI in affected mice is linked to ER stress rather than dysfunction of secreted amelogenin. An unexpected finding was that the 32 kDa enamelin (much lauded in the literature as a functional species) may be unique to pig amelogenesis and its functional significance is therefore debateable In summary, the mechanism driving AI in these mice is associated with intracellular ER stress. Extracellular dysfunction of mutated enamel proteins has been the focus of AI research but the involvement of ER stress provides additional therapeutic options for treating AI.