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 Ͻ0.2% 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 ␤-protein (A␤)-induced neuronal compromise significantly increased the quantity of all forms of extracellular tau, but the presence of A␤ 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.

BackgroundExosomes, 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.ResultsWe 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 PrPC rather than Aβ proteolysis.ConclusionsThese data suggest that exosomes can counteract the inhibitory action of Aβ, which contributes to perpetual capability for synaptic plasticity.

IntroductionAmyloid 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.MethodsWe tested four antibody pairs and chose one pair that was particularly sensitive, using 1C22, our new oligomer-selective monoclonal antibody, for capture. We applied this new assay to extracts of human brain and CSF.ResultsA combination of 1C22 for capture and 3D6 for detection yielded an Erenna immunoassay with a lower limit of quantification of approximately 0.15 pg/ml that was highly selective for oligomers over monomers and detected a wide size-range of oligomers. Most CSFs we tested had detectable oligomer levels but with a large overlap between AD and controls and a trend for higher mean levels in mild cognitive impairment (MCI) than controls.ConclusionAβ oligomers are detectable in most human CSFs, but AD and controls overlap. MCI CSFs may have a modest elevation in mean value by this assay.Electronic supplementary materialThe online version of this article (doi:10.1186/s13195-015-0100-y) contains supplementary material, which is available to authorized users.

We present the design, synthesis, and functional evaluation of peptide-based fluorescent constructs for wavelength-ratiometric biosensing of a protein analyte. The concept was shown using the high-affinity model interaction between the 18 amino acid peptide pTMVP and a recombinant antibody fragment, Fab57P. pTMVP was functionalized in two different positions with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive fluorophore with a two-band emission. The equilibrium dissociation constant of the interaction between pTMVP and Fab57P was largely preserved upon labeling. The biosensor ability of the labeled peptide constructs was evaluated in terms of the relative intensity change of the emission bands from the normal (N*) and tautomer (T*) excited-state species of the fluorophore ( I(N*)/I(T*)) upon binding of Fab57P. When the peptide was labeled in the C terminus, the I(N*)/I(T*) ratio changed by 40% upon analyte binding, while labeling close to the residues most important for binding resulted in a construct that completely lacked ratiometric biosensor ability. Integrated biosensor elements for reagentless detection, where peptides and ratiometric fluorophores are combined to ensure robustness in both recognition and signaling, are expected to become an important contribution to the design of future protein quantification assays in immobilized formats.

No abstract

No abstract

We report the formation of a non-native, folded state of human IgG4-Fc induced by a high temperature at neutral pH and at a physiological salt concentration. This structure is similar to the molten globule state in that it displays a high degree of secondary structure content and surface-exposed hydrophobic residues. However, it is highly resistant to chemical denaturation. The thermally induced state of human IgG4-Fc is thus associated with typical properties of the so-called alternatively folded state previously described for murine IgG, IgG-Fab, and individual antibody domains (V(L), V(H), C(H)1, and C(H)3) under acidic conditions in the presence of anions. Like some of these molecules, human IgG4-Fc in its alternative fold exists as a mixture of different oligomeric structures, dominated by an equilibrium between monomeric and heptameric species. Heating further induces the formation of fibrous structures in the micrometer range.

from human brain extracts. Our ongoing work includes testing the hypothesis that post-translational modifications and co-associated proteins may contribute to the development and accumulation of toxic oligomer assemblies which are specific to demented patients versus nondemented patients with similar levels of A b oligomers.