The amyloid β (Aβ)-protein and microtubule-associated protein, tau, are the major components of the amyloid plaques and neurofibrillary tangles that typify Alzheimer's disease (AD) pathology. As such both Aβ and tau have long been proposed as therapeutic targets. Immunotherapy, particularly targeting Aβ, is currently the most advanced clinical strategy for treating AD. However, several Aβ-directed clinical trials have failed, and there is concern that targeting this protein may not be useful. In contrast, there is a growing optimism that tau immunotherapy may prove more efficacious. Here, for the first time, we studied the effects of chronic administration of an anti-tau monoclonal antibody (5E2) in amyloid precursor protein transgenic mice. For our animal model, we chose the J20 mouse line because prior studies had shown that the cognitive deficits in these mice require expression of tau. Despite the fact that 5E2 was present and active in the brains of immunized mice and that this antibody appeared to engage with extracellular tau, 5E2-treatment did not recover age-dependent spatial reference memory deficits. These results indicate that the memory impairment evident in J20 mice is unlikely to be mediated by a form of extracellular tau recognized by 5E2. In addition to the lack of positive effect of anti-tau immunotherapy, we also documented a significant increase in mortality among J20 mice that received 5E2. Because both the J20 mice used here and tau transgenic mice used in prior tau immunotherapy trials are imperfect models of AD our results recommend extensive preclinical testing of anti-tau antibody-based therapies using multiple mouse models and a variety of different anti-tau antibodies.

Amyloid β-protein oligomers play a key role in Alzheimer’s disease (AD), but well-validated assays that routinely detect them in cerebrospinal fluid (CSF) are just emerging. We sought to confirm and extend a recent study using the Singulex Erenna platform that reported increased mean CSF oligomer levels in AD.

Recent evidence suggests that tau aggregation may spread via extracellular release and subsequent uptake by synaptically connected neurons, but little is known about the processes by which tau is released or the molecular forms of extracellular tau. To gain insight into the nature of extracellular tau, we used highly sensitive ELISAs, which, when used in tandem, are capable of differentiating between full-length (FL) tau, mid-region-bearing fragments, and C-terminal (CT) fragments. We applied these assays to the systematic study of the conditioned media of N2a cells, induced pluripotent stem cell-derived human cortical neurons, and primary rat cortical neurons, each of which was carefully assessed for viability. In all three neuronal models, the bulk of extracellular tau was free-floating and unaggregated and <0.2% was encapsulated in exosomes. Although most intracellular tau was FL, the majority of extracellular tau was CT truncated and appeared to be released both actively by living neurons and passively by dead cells. In contrast, only a small amount of extracellular tau was aggregation-competent tau (i.e., contained the microtubule-binding regions) and this material appears to be released solely due to a low level of cell death that occurs in all cell culture systems. Importantly, amyloid β-protein (Aβ)-induced neuronal compromise significantly increased the quantity of all forms of extracellular tau, but the presence of Aβ before detectable cell compromise did not increase extracellular tau. Collectively, these results suggest that factors that induce neuronal death are likely to be necessary to initiate the extracellular spread of tau aggregation. SIGNIFICANCE STATEMENT:Recent studies suggest that the transfer of tau between neurons underlies the characteristic spatiotemporal progression of neurofibrillary pathology. We searched for tau in the conditioned medium of N2a cells, induced pluripotent stem cell-derived human cortical neurons, and primary rat cortical neurons and analyzed the material present using four different tau ELISAs. We demonstrate that the majority of tau released from healthy neurons is C-terminally truncated and lacks the microtubule-binding region (MTBR) thought necessary for self-aggregation. A small amount of MTBR-containing tau is present outside of cells, but this appears to be solely due to cell death. Therefore, if propagation of tau aggregation is mediated by extracellular tau, our findings suggest that neuronal compromise is required to facilitate this process.

Humanized anti-Abeta murine monoclonal IgGs (mAbs) generated against linear or conformational epitopes are investigational therapeutics that are being developed for Alzheimer's disease (AD). Such antibodies may have pan-Abeta conformer reactivity or preferential binding to a specific peptide conformer (aggregates or monomer) due to a unique exposed surface(s) and/or avidity effects (bivalent binding). To advance understanding on the Abeta conformer reactivity of clinically tested mAbs, we used a battery of solid- and solution-phase assays to compare the reactivities of murine forms of Bapineuzumab (3D6, N-terminal) and Solanezumab (266, mid-region) with our novel lead Abeta aggregate-preferring mAb, 1C22. Aggregate-preferring anti-Abeta IgGs were generated by immunizing mice with prefibrillar assemblies formed from disulfide cross-linked Abeta dimer (AbetaS26C)2. Hybridomas secreting anti-Abeta mAbs were produced by conventional fusion methods, and a lead aggregate-preferring mAb identified by solid phase. Sandwich and competition ELISAs; surface Plasmon resonance (SPR); microscale thermophoresis, and in vitro and ex vivo immunohistochemistry were used to compare forms of Abeta recognized by mAbs 3D6, 266, and our lead mAb 1C22. Our initial screen of 19 novel, in-house generated anti-Abeta mAbs utilized plate-immobilized Abeta conformers to identify antibodies that preferentially bound to protofibrils. MAb 1C22 was identified as a lead antibody candidate with nM binding to protofibrils and uM reactivity with Abeta monomer. In all other assays 1C22 showed a strong preference for protofibrils and fibrils - a preference that was largely driven by bivalent binding (avidity effects). Although less pronounced, 3D6, 6E10, and 4G8 also preferentially bound protofibrils, whereas mAb 266 bound strongest to Abeta monomer. Indeed, mAb 266 only bound to surface-immobilized Abeta aggregates under certain conditions. The pros and cons of these mAbs as therapeutic and diagnostic tools, and the methods used to assess their properties will be discussed.

Background Exosomes, small extracellular vesicles of endosomal origin, have been suggested to be involved in both the metabolism and aggregation of Alzheimer’s disease (AD)-associated amyloid β-protein (Aβ). Despite their ubiquitous presence and the inclusion of components which can potentially interact with Aβ, the role of exosomes in regulating synaptic dysfunction induced by Aβ has not been explored. Results We here provide in vivo evidence that exosomes derived from N2a cells or human cerebrospinal fluid can abrogate the synaptic-plasticity-disrupting activity of both synthetic and AD brain-derived Aβ. Mechanistically, this effect involves sequestration of synaptotoxic Aβ assemblies by exosomal surface proteins such as PrP C rather than Aβ proteolysis. Conclusions These data suggest that exosomes can counteract the inhibitory action of Aβ, which contributes to perpetual capability for synaptic plasticity.