In this study, we developed novel methods to understand which of several simultaneously existing in situ epitopes of diffuse mHtt best predicts neurotoxicity. We used a series of antibodies to distinguish species of htt in situ and then automated microscopy with survival analysis to determine whether any of them predicted toxicity in a primary striatal neuron model of HD. Among four antibodies we compared (3B5H10, MW1, MW7 and EM48), the newly developed mAb 3B5H10 bound a species of htt that best predicted neurodegeneration.
The epitope recognized by 3B5H10 is contained within the polyQ stretch of mHtt but disappears as mHtt aggregates into IBs. The epitope exists in both full-length and an exon1 fragment of mHtt but is negligibly present in wt-htt. Further, the epitope also exists in mutant forms of other polyQ-containing proteins that cause neurodegeneration, including the androgen receptor, atrophin, and ataxin-3. Finally, we determined that the epitope exists in certain conformational folds of polyQ associated with low molecular weight htt species (monomers and possibly very small oligomers).
We considered whether 3B5H10 is capable of binding oligomers of mHtt even though it preferentially binds a monomeric species. While 3B5H10 strongly stains the diffuse mHtt fraction of neurons, we did not detect oligomers in this fraction by FRET or an α-oligomer antibody. Slot blot, agarose gel electrophoresis, AFM, and DLS (, Supplementary Figs. 9–10
) confirmed that the epitope recognized by 3B5H10 is present in a very low molecular weight species of htt (likely monomer and possibly small oligomer). Chemical cross-linking experiments () confirmed the epitope 3B5H10 recognizes is present in monomers and, to a lesser degree, in very small oligomers of htt. 3B5H10’s tendency to bind monomers over oligomers was further supported by results from size-exclusion chromatography (), sedimentation equilibrium analytical ultracentrifugation (), and small-angle X-ray scattering (unpublished observations).
A recent study on the formation of different conformations of mHtt found that 3B5H10 may bind a conformation of amyloid formed in vitro
This structure, unlike amyloid formed in vitro
at 37°C, has a loop/turn organization that exposes loose hairpins of polyQs. Additionally, certain biochemical purification techniques may facilitate de novo
exposure of the 3B5H10 epitope on oligomers35
, analogous to unmasking of the 3B5H10 epitope upon aggressive antigen retrieval of HD animal model brains (Supplementary Fig. 4 online
). Combined with our data showing very faint 3B5H10 staining of cross-linked small htt oligomers (), we conclude that under certain in vitro
conditions or under certain biochemical and immunohistochemical methods, the conformation of polyQ recognized by 3B5H10 may appear in aggregated species of htt.
The ability of 3B5H10 to dissociate pre-formed oligomers and fibrils into monomeric htt (, Supplementary Fig. 10 online
) was surprising. We are aware of only one other α-htt antibody that demonstrates this property, MW77
. However, MW7 appears to dissolve pre-formed fibrils into AFM-detectable species with dimensions that are consistent with an oligomer rather than a monomer. We speculate that 3B5H10 may promote oligomer and fibril dissociation by sequestering monomers that could be dynamically associating and dissociating with oligomers or fibril ends36,37
Since 3B5H10 and MW1 both bind polyQ expansions in a length-dependent manner and preferentially recognize diffuse mHtt in situ4,33
, it is surprising that only 3B5H10 binding predicts neurodegeneration by multivariate Cox analysis (see legend for Supplementary Fig. 5 online
). One explanation is that MW1 and 3B5H10 bind mostly distinct conformers of mHtt, only one of which may have prognostic value. Previous studies demonstrated that MW1 recognizes expanded polyQ as a “linear lattice”21,33
. In such a model, the antibody binds weakly to a relatively unstructured epitope of wt polyQ. As the length of the polyQ stretch approaches that associated with disease, the unstructured epitope repeats. Since antibodies are bivalent, the presence of two epitopes in tandem results in dramatically increased binding by MW1, due to increased avidity (Supplementary Fig. 13 online
). In contrast, our results and our unpublished structural data demonstrate that 3B5H10 recognizes a compact, structured epitope of polyQ that is minimally present in wt-htt and is exposed or created in mHtt. Thus, rather than preferentially binding expanded polyQ via increased avidity, 3B5H10 demonstrates a strong affinity for mutant polyQ. Supporting these conclusions, MW1 Fab forms a 3:1 complex33
with the exact version of htt (Thio-Httex1
) that we found forms a 1:1 complex with 3B5H10 Fab (, Supplementary Fig. 13 online
). Thus, the “linear lattice” epitope that MW1 recognizes is no larger than 13 glutamines, consistent with crystallographic studies of MW1 complexed with a polyQ peptide21
. Also supporting these conclusions, the Fab of MW1, which is monovalent and therefore can not bind its target through an avidity mechanism, loses most of its preference for mutant polyQ over wt polyQ33
. In contrast, the Fab of 3B5H10 retains a preference for mutant polyQ (, Supplementary Fig. 13 online
). These observations, combined with our structural studies (unpublished data), suggest that low molecular weight species of mHtt may exist in more than one conformation and that the one recognized by 3B5H10 might be toxic or closely related to a toxic species ()38–40
Figure 7 The “linear lattice” versus “emergent conformation” hypotheses for expanded polyQ conformation. The linear lattice model posits that a relatively unstructured epitope of polyQ in wt-htt repeats itself as the polyQ stretch (more ...)
A burgeoning body of work in polyQ disease research suggests that culprit proteins may exert their toxic gain-of-function by enhanced native activity (especially via native protein-protein interactions41,42
). Since our exon1 model of HD, by definition, does not encompass all native function of full-length htt, we may miss pathogenic events that rely on native activity of htt outside exon1. While technical barriers prohibited us from designing a full-length primary culture model of HD for these experiments, we have previously extensively validated our exon1 model as a faithful model of HD43
. Further, we demonstrate in this paper the ability of 3B5H10 to recognize full-length mHtt, ataxin-3, and androgen receptor, thereby establishing the presence of the predictive epitope in full-length protein. Finally, we note that the majority of known htt interactors bind to the N-terminus of htt44–46
and, in some cases, this has been defined even more narrowly to be the exon1 region46
. Thus, we favor the hypothesis that the 3B5H10 conformer of httex1
still mediates toxicity at least partially through native activity, including enhanced interactions with some exon1-interactors and diminished interactions with other exon1-interactors.
We previously found that IB formation is associated with improved neuronal survival10
, and in our current studies, we discovered that IB formation leads to a substantial loss of intraneuronal 3B5H10 binding. In contrast, IB formation does not lead to a loss of EM48 or MW7 binding (). Since 3B5H10 binds a species of mHtt that strongly predicts death (better than the epitopes recognized by EM48 or MW7, for example), IB formation might be protective by preferentially reducing, masking, or refolding the 3B5H10 epitope (Supplementary Fig. 14 online
The novel methodology we employed may be broadly applicable to the study of diseases associated with protein malfolding. Elucidating toxic species of aggregation prone proteins is difficult because these species may be rare, their existence may depend on endogenous protein interactions that defy biochemical purification, and the tools to study protein conformation in situ are limited. We showed that combining the use of conformation-specific antibodies and automated imaging with longitudinal analysis provides a way to probe protein conformation in situ and elucidate the prognostic significance of one conformer in the context of others. We expect that the better a conformer predicts neurodegeneration, the more tightly it is linked to toxicity and pathogenesis.