Anti-A miniantibodies suppress A42 neurotoxicity in flies. Pedro Fernandez-Funez^1,2,3, Swati Khare¹, Krishanu Mathur¹, Alfonso Martin-Peņa¹, Diego Rincon-Limas^1,2. 1) Dept Neurology; 2) Dept Neuroscience; 3) Genetics Institute and Center for Translational Research on Neurodegenerative Diseases; University of Florida, Gainesville, FL.

   Alzheimers disease (AD) is an incurable neurodegenerative disorder characterized by irreversible cognitive decline. Soluble assemblies of the Amyloid- (A42) peptide are the leading neurotoxic agents in AD pathogenesis; thus, strategies that target A42 are likely to slow or revert the disease. Passive immunotherapy is a promising approach in AD; sadly, intravenous delivery of a full size anti-A antibody failed to improve cognition recently in human clinical trials. There is still hope, though, that single chain variable fragments (scFv) may demonstrate better efficacy due to their higher penetration in the brain. We want to use Drosophila as a platform to test the in vivo activity of new anti-A42 scFvs. As proof-of-principle, we generated flies expressing scFv-A9 and -213, two scFvs known to bind A42 and reduce plaque formation in mice. Since the AD mice do not exhibit significant neuronal loss, we examined the neuroprotective effect of these scFvs in our Drosophila model expressing human A42. We found that both scFv-A9 and -213 partially suppressed the A42 phenotype, although scFv-A9 worked better. Interestingly, co-expression of both scFv-A9 and -213 resulted in even better suppression of A42 neurotoxicity, suggesting a synergistic activity from binding to the two epitopes. Expression of each scFvs significantly reduced the number of apoptotic cells induced by A42. But the combination of both miniantibodies potently inhibited Casp3 activation to levels comparable to normal eyes. We also found that the scFvs do not affect the levels of total A42 by Western blot or the accumulation of amyloid fibers by thioflavin-S staining. Thus, the scFvs mediate their protective activity by preventing the interaction of A42 with cellular targets, not by promoting A42 degradation. These results demonstrate that secreted scFvs work well in flies and, hence, can be used to screen for highly effective scFvs or combinations thereof against A42.