Alzheimer's disease (AD) is a neurodegenerative disorder that nowadays affects more than 40 million people worldwide and it is predicted to exponentially increase in the coming decades. Because no curative treatment exists, research on the pathophysiology of the disease, as well as the testing of new drugs, are mandatory. For these purposes, animal models constitute a valuable, although perfectible tool. This review takes a tour through several aspects of mouse models of AD, such as the generation of transgenic models, the relevance of the promoter driving the expression of the transgenes, and the concrete transgenes used to simulate AD pathophysiology. Then, transgenic mouse lines harboring mutated human genes at several loci such as APP, PSEN1, APOEɛ4, and ob (leptin) are reviewed. Therefore, not only the accumulation of the Aβ peptide is emulated but also cholesterol and insulin metabolism. Further novel information about the disease will allow for the development of more accurate animal models, which in turn will undoubtedly be helpful for bringing preclinical research closer to clinical trials in humans.

The intracellular deposition of amyloid-␤ (A␤) peptides has been described in the brains of both Alzheimer's disease (AD) patients and animal models. A correlation between the intracellular amyloid burden and neurodegeneration has recently been reported in a triple-transgenic AD (3xTg-AD) murine model. In the present study, we assessed the effect of scFv-h3D6, an anti-A␤ single-chain variable fragment (scFv) derived from the antibody bapineuzumab, on amyloid pathology in 5-month-old 3xTg-AD female mice, focusing on intracellular A␤ clearance, neuronal survival, and functional abilities. We also examined neuroinflammation and the histology of peripheral organ samples to detect any adverse effects. A single intraperitoneal injection of scFv-h3D6 dramatically reduced intracellular A␤ burden in the deep layers of the cerebral cortex, pyramidal cells layer of the hippocampus, and basolateral amygdalar nucleus. The treatment prevented neuronal loss in the hippocampus and amygdala, while neither astrogliosis nor microgliosis was induced. Instead, an increase in the size of the white pulp after the treatment indicated that the spleen could be involved in the clearance mechanism. Although the treatment did not ameliorate behavioral and psychological symptoms of dementia-like symptoms, the results of cognitive testing pointed to a noticeable improvement in spatial memory. These findings indicated that the mechanism underlying the therapeutic effect of scFv-h3D6 was the clearance of intracellular A␤, with subsequent prevention of neuronal loss and amelioration of cognitive disabilities. The treatment was safe in terms of neuroinflammation and kidney and liver function, whereas some effects on the spleen were observed.

ScFv-h3D6 has been shown as an efficient therapy in the 3xTg-AD mouse model of Alzheimer’s Disease. Because one of the major bottlenecks for the therapeutic uses of proteins produced in Escherichia coli is their potential contamination with endotoxins, LPS were extensively removed by a rather low-efficient, expensive, and time-consuming purification step. In addition, disulfide scrambling is favored in the reducing bacterial cytoplasm albeit the use of reductase deficient strains. To overcome these hurdles, as well as to improve the yield, the yeast Pichia pastoris, an endotoxin-free host system for recombinant protein production, has been used to produce scFv-h3D6, both in flask and in a fed-batch bioreactor. Comparison of the thermal stability of the obtained protein with that from E. coli showed no differences. Opposite to the case of the protein obtained from E. coli, no disulfide scrambled conformations or LPS traces were detected in that produced in P. pastoris. Cytotoxicity assays in SH-SY5Y neuroblastoma cell-cultures demonstrated that proteins from both expression systems were similarly efficient in precluding Aβ-induced toxicity. Finally, the 3xTg-AD mouse model was used to test the therapeutic effect of both proteins. Quantification of Aβ levels from cortex and hippocampus protein extracts by ELISA, and Aβ-immunohistochemistry, showed that both proteins reduced Aβ burden. This work demonstrates that scFv-h3D6 obtained from P. pastoris shows the same benefits as those already known for that obtained from E. coli, with multiple advantages in terms of recombinant production and safety.

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