ABSTRACT
Here we present the ascidian Ciona intestinalis as an alternative invertebrate system to study Alzheimer's disease (AD) pathogenesis. Through the use of AD animal models, researchers often attempt to reproduce various aspects of the disease, particularly the coordinated processing of the amyloid precursor protein (APP) by alpha-, beta- and gamma-secretases to generate amyloid beta (Abeta)-containing plaques. Recently, Drosophila and C. elegans AD models have been developed, exploiting the relative simplicity of these invertebrate systems, but they lack a functional Abeta sequence and a beta-secretase ortholog, thus complicating efforts to examine APP processing in vivo. We propose that the ascidian is a more appropriate invertebrate AD model owing to their phylogenetic relationship with humans. This is supported by bioinformatic analyses, which indicate that the ascidian genome contains orthologs of all AD-relevant genes. We report that transgenic ascidian larvae can properly process human APP(695) to generate Abeta peptides. Furthermore, Abeta can rapidly aggregate to form amyloid-like plaques, and plaque deposition is significantly increased in larvae expressing a human APP(695) variant associated with familial Alzheimer's disease. We also demonstrate that nervous system-specific Abeta expression alters normal larval behavior during attachment. Importantly, plaque formation and alterations in behavior are not only observed within 24 hours post-fertilization, but anti-amyloid drug treatment improves these AD-like pathologies. This ascidian model for AD provides a powerful and rapid system to study APP processing, Abeta plaque formation and behavioral alterations, and could aid in identifying factors that modulate amyloid deposition and the associated disruption of normal cellular function and behaviors.
Subject(s)
Alzheimer Disease/etiology , Disease Models, Animal , Urochordata/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amino Acid Sequence , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/metabolism , Animals , Animals, Genetically Modified , Behavior, Animal , Conserved Sequence , Embryo, Nonmammalian/metabolism , Embryo, Nonmammalian/pathology , Humans , Larva , Molecular Sequence Data , Plaque, Amyloid/pathology , Protein Processing, Post-TranslationalABSTRACT
Two customized electroporators were specifically designed for creating transgenic ascidian embryos. These electroporators were simple to build, inexpensive, and produced transgenic embryos with efficiencies that equaled or rivaled commercially available machines. A key design feature of these machines resulted in the generation of consistent electroporation pulses providing repeatability between experiments. These devices were used to optimize experimental parameters allowing for the creation of transient transgenic embryos with predictable patterns of mosaic transgene expression. We used these new electroporators to examine the expression of two different fluorescent protein reporter genes with regard to embryonic cell lineage. In general, transgene expression followed the embryonic cell lineage and coelectroporated transgenes were always expressed in the same embryonic cells. Our analysis also indicated that, during development, transgenes could be lost from embryonic cells, suggesting that transgenes may be present in extrachromosomal arrays, as has been observed in other organisms. Our new electroporator designs will allow ascidian researchers to inexpensively produce transgenic ascidians and should prove useful for adapting the electroporation technique to other marine embryo systems.
