A debate rages over the relevance of nonmammalian animal models to what is a uniquely human disease. Much has been learned about the basic biology of AD mechanisms from nonmammalian systems. Biochemical processes tend to follow general precepts that are comparable in simple and complex organisms. The yeast Saccharomyces cerevisiae allows the use of powerful classical and molecular genetic tools, although this organism lacks a nervous system. The roundworm Caenorhabditis elegans and the fruit fly Drosophila melanogaster are well-studied animals that are also genetically accessible. Both feature relatively simple, stereotyped nervous systems to analyze biochemical pathways and cell function. C. elegans (Rankin, 2004) and Drosophila (Rohrbough et al., 2003) display certain aspects of learning behavior that have been dissected genetically. The genomes of all these organisms have been sequenced, so mammalian and human gene homologs can be readily identified. Short generation times shrink the timescale of experiments with these organisms, even for elderly individuals.
While possessing a number of advantages for studying specific instances of biochemical pathways, mechanisms, or physiological subsystems, there remain significant differences in complexity and organization between invertebrate and mammalian nervous systems that limit the simpler organisms as AD models. Although these organisms have been used as models of aging, and there are certain similarities, questions remain about the equivalence of two months of life in fruit flies to 80 years in humans.
Nonmammalian organism models Drosophila and C. elegans provide both powerful genetic tools and a sufficiently complex behavioral repertoire to be able to detect alterations in brain function (Driscoll and Gerstbrein, 2003). They possess homologs to the human genes that are linked to familial AD. Unfortunately, neither the fly nor worm ftAPP contains the amyloidogenic sequence, and thus natural AD ft-amyloid models are not available. The fly, in particular, has been useful for studies in AD, in Huntington's disease (HD), and in Parkinson's disease (PD) when the human familial disease transgenes are expressed leading to neurodegeneration. By a judicious choice of promoters, the mutant gene is directed to an easily observed neural tissue, the compound eye, in which neurodegeneration can be easily monitored.
In C. elegans expression of human Aft(1-42) in muscle leads to toxic effects (Fonte et al., 2002). By searching for suppressor genes for the toxic phenotype in both flies and worms, potential pathways for countering the neurodegeneration have been identified. An example is the hsp70 chaperone protein, which reduces the accumulation of misfolded proteins. Mutation of negative regulators of hsp70 activity also suppresses the toxic phenotype. Toxicity of misfolded proteins linked to AD, PD, and HD in these organisms is ameliorated by increased hsp70 activity. However, implementing a therapeutic strategy of increased hsp70 levels is likely to be fraught with difficulties due to the multiple roles of hsp70 in cell biology and the consequences of a sustained stress response.
Ex-vivo models In this category we include both isolated cell culture and tissue slice organotypic culture systems that approximate various aspects of AD. Ex vivo models are a step up in complexity—and hopefully disease relevance—from in vitro assays of biochemical components and biophysical processes. A variety of cell lines have been used via transfection of appropriate genes to study pAPP metabolism and to work out the consequences of the familial AD mutations in pAPP and the presenilins. Under the premise that many of the events are similar in all cells, even ones not thought to be altered in AD, these systems have been used to probe the cell biology of the amyloid cascade. Overall correspondence of results with those from neuronally derived cell lines is cited as evidence for the validity of these models. Indeed, the link between mutant presenilins and the increased production of Ap in familial AD was accurately predicted from cell culture models.
Major caveats to blanket acceptance of the isolated cell systems as models of AD are that they are mostly transformed, relatively undifferentiated cells, often of nonneuronal origin or mixed neuronal-glial origin. Those cell lines of neuronal origin are frequently derived from the peripheral nervous system, which is spared in AD. Cultured cells also do not manifest the multiple connections or the extreme polarity and long-distance transport requirements of central neurons that are most affected in AD. Neurons cultured from embryonic or fetal brain regions are more representative of differentiated neurons, although they are mixtures of different kinds of neurons, in early stages of differentiation, that are removed from the surroundings in which they developed. There is much discussion about how similar these neurons are to in situ neurons because of the selection pressures in vitro. Fetal neurons from some brain regions survive to various degrees, while fully differentiated neurons from postnatal animals rarely are successfully cultured.
Organotypic brain-slice cultures are routinely used for neurotransmitter electrophysiology studies. The connectivity of various regions is more or less intact, depending on how the slices are made. Advantages are that the cells maintain their local contacts and are more highly differentiated than are embryonic cultures. Disadvantages are that they are labor- and resource-intensive, generally not long-term, and are difficult to prepare successfully from older animals. These systems have been useful for distinguishing neuronal toxicity of soluble monomeric, oligomeric and fibrillar p-amyloid species (Lacor et al., 2004) and the effects of Ap on electrophysiology, such as blocking LTP but not LTD (Walsh and Selkoe, 2004).
Cerebrovascular smooth muscle cells cultured from human, canine, and pAPP-transgenic mouse (Tg2576) leptomeninges actively produce Ap peptides, secreting them into the medium and depositing them intra-cellularly and extracellularly (Frackowiak et al., 2003; Frackowiak et al., 2005). This is the only cell system that deposits Ap in culture. The smooth muscle cells are susceptible to Ap toxicity, particularly to a mutant peptide, Ap E22Q, that produces a selective cerebrovas-cular smooth muscle amyloidosis with little parenchymal involvement in humans and in mouse models. This is a highly relevant system since these cells are involved in cerebral p-amyloid angiopathy (CAA), which is seen in virtually all AD patients and can lead to intracerebral hemorrhages.
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