| The broad objective of this project is to provide supporting evidence for the use of the ascidians Ciona intestinalis and Ciona savignyias new model organisms (MOs) for Alzheimer's disease (AD) basic research. Amyloid Precursor Protein (APP), is an integral membrane protein strongly implicated in the pathogenesis of AD. Aberrant processing of APP generates the aggregating peptide amyloid beta (Abeta) and is thought to be a causative factor in AD development and progression. APP orthologues have been identified in many vertebrate and invertebrate species. Recently, human APP (hAPP) proteolytic processing has been shown to be conserved in transgenic intestinalis expressing the hAPP gene. An APP gene prediction has been made in C.savignyi, the closest genetic relative to C. intestinalis, currently there is no known C.intestinalis APP gene model, (CiAPP).The homologous C.savignyi APP(CsAPP) was identified from the Ghost Database and cloned by conventional methods. The cloned CsAPP gene was used to generate an RNA probe for temporal and spatial expression pattern determination. Hypothesized high expression in the central nervous system (CNS) was assessed by whole mount in situ hybridization (WISH), CNS expression was observed at 10, 12, 14 and 15 hours post fertilization in C.savignyi larvae. To confirm this result a putative 4kb promoter region was identified and cloned into a GFP reporter construct. To determine if human AICD (hAICD),translocates to the nucleus with the putative function as a transcriptional activator, two mutant hAPP, fluorescent protein reporter constructs were electroporated into C. intestinalis embryos to visualize the subcellular localization of hAICD. Although infrequent, at 18hrs post fertilization, hAICD was observed localized in some nuclei. These results are a first step towards the characterization of an ascidian APP homolog, including its expression pattern, transcriptional promoter information and role in intracellular signaling and will further help to establish and validate the ascidians C.savignyi and C. intestinalis as novel MOs in AD research. |
