Aß “Affibodies:” A Novel Route to Comprehensive Clearance?

To test the effects of  Z_Aβ3 on Aß aggregatess and neurotoxicity in vivo,  Swedish and English collaborators used Drosophila lines transgenically expressing either wild-type Aß42, E22G (the preferrentially oligomer-forming “Arctic” Aß mutation first identified in a Swedish familial AD proband), or the relatively benign, non-aggregative isoform Aß40. These were then crossed with strains transgenic for Z_Aβ3; for 2 copies of Z_Aβ3 connected head-to-tail ((Z_Aβ3)₂) to facilitate formation of the Z_Aβ3 dimer; or for overexpression of the wild-type Z domain alone (as controls). The resulting hybrids were crossed onto strains bearing drivers to ensure coexpression of the transgenes in brain neurons or photoreceptors.(9)

Transgenic Aß42 is severely neurotoxic to Drosophila, reducing lifespan from 38 days in controls to 28 d (WT Aß42) or 9 d (E22G). E22G neurotoxicity was also evident in the eye, where expression led to abnormal photoreceptor morphology. Z_Aβ3 substantially rescued fly lifespan against the expressed Aß, to 32 and 20 d, respectively. (Z_Aβ3)₂ proved yet more effective: it fully rescued lifespan from the effects of WT Aß42 (to 40 d) and more fully normalized it against the effects of E22G (31 d), while substantially protecting photoreceptor morphology. Aß40 was harmless to the flies, and lifespan of TG-Aß40 Drosophila was unaffected by coexpression of affibodies. 

Affibody clearance of Aß E22G was also evident, with Aß levels being high in E22G-TG  Drosophila but considerably reduced in flies coexpressing Z_Aβ3 and indetectable with coexpression of (Z_Aβ3)₂. That the mechanism of Aß reduction was not due to effects on Aß production was demonstrated by the lack of a significant effect on neuronal E22G expression. More intriguingly, the reduction in Aß was also not accompanied by a concomitant buildup in SDS-stable  Z_Aβ3-Aß complexes, as demonstrated by a lack of appearance of liberated Aß following treatment with guanidinium chloride to dissociate such complexes. Rather, the absolute, total level of Aß in E22G-TG Drosophila was ~75% lower in flies coexpressing  Z_Aβ3 monomer, and by 97±3% with coexpressed  (Z_Aβ3)2, relative to levels in controls coexpressing inert Z domain.(9)

Reductions in monomeric Aß were accompanied by equivalent reductions in Aß fibrils in vivo, and in vitro studies showed that addition of affibody to Aß42 or -40 isoforms exerts equivalent effects on the kinetics of fibril formation as do equivalent reductions in initial levels of Aß monomer in the medium, implying that the reduction in fibril levels is primarily due to the initial binding of affibody to Aß monomer. Yet addition of excessive affibody later in the aggregation reaction process also arrests further fibril formation and even leads to a very gradual reduction in levels of existing fibrils, accompanied by the appearance of affibody-bound Aß monomers; this result suggests some ability to bind Aß monomers within fibrillar and protofibrillar Aß structures, and to slowly contribute to their dissociation (although at rates so low as to be unlikely to occur in situ in the brain).(9)

On the other hand, similar studies not only revealed a similar ability of Z_Aβ3 to prevent the formation of Aß oligomers from monomeric Aß, but also a physiologically plausible destabilization and dissolution of existing oligomers.(9)

The sum of the results suggest that initial sequestration of Aß monomers by Z_Aβ3 — as free Aß molecules or as constituents of oligomeric and perhaps protofibrillar or even fibrillar Aß — is later followed by degradation of affibody-bound Aß in vivo. The authors note that the mechanism underlying any such effect cannot involve clearance of Aß species by anti-Aß antibodies induced in the flies through activation of an adaptive immune response, as Drosophila do not have an adaptive immune system. Rather, they suggest that affibody-bound Aß monomers are subsequently degraded by endogenous biological mechanisms, such as proteasomal or lysosomal breakdown within neurons, or by phagocyes following an hypothetical exocytosis.(9) If correct, the rate of such degradation could in principle be enhanced if necessary by fortification of the lysosome with novel hydrolases (lysoSENS) targeting residual lysosomal Aß accumulations  or any specific degradation-resistant aggregates formed following lysosomal uptake and storage.(13-15)

Obviously, these studies, while promising, are very preliminary. Drosophila is not an ideal model organism in which to test the effects of Aß and its clearance in vivo, and neuronal Z_Aβ33 coexpression studies can only be taken as suggestive of the possible effects of affibody delivered by systemic injection. Moreover, there remain many open questions and concerns about the timecourse and mechanism of Aß disappearance, such as whether Z_Aβ33 can indeed clear out existing accumulations of Aß oligomers and fibrils in aging human brains, and whether a mechanism apparently largely reliant on sequestration of free monomers might interfere with any putative physiologic role of Aß in the brain(10-12).

But a novel route toward Aß removal that does not rely on the immune system, and that offers the promise of removal of a wide range of Aß aggregates but especially of oligomeric species, is a promising new development in the field. Indeed, while speculation from this preliminary result cannot be given much weight, under a best-case scenario one can envision the opening of an entirely new approach to therapeutic clearance not only of Aß, but of a range of other disease- and aging-associated intracellular and extracellular aggregates. The broad outlines of the line of investigation required to turn these preliminary results into a therapy for brain aging and AD is reasonably clear; if its potential is realized clinically, then affibody-based therapeutics may ultimately be used to prevent and even reverse age-related diseases ranging from other forms of neurodegeneration, to senile cardiac amyloidosis, to age-related macular degeneration, to atherosclerosis. We await further revelations from these investigators with tempered optimism.