Comparative study of naturally occurring huntingtin fragments in Drosophila points to exon 1 as the most pathogenic species in Huntington's disease.

Although Huntington's disease is caused by the expansion of a CAG triplet repeat within the context of the 3144-amino acid huntingtin protein (HTT), studies reveal that N-terminal fragments of HTT containing the expanded PolyQ region can be produced by proteolytic processing and/or aberrant splicing. N-terminal HTT fragments are also prevalent in postmortem tissue, and expression of some of these fragments in model organisms can cause pathology. This has led to the hypothesis that N-terminal peptides may be critical modulators of disease pathology, raising the possibility that targeting aberrant splicing or proteolytic processing may present attractive therapeutic targets. However, many factors can contribute to pathology, including genetic background and differential expression of transgenes, in addition to intrinsic differences between fragments and their cellular effects. We have used Drosophila as a model system to determine the relative toxicities of different naturally occurring huntingtin fragments in a system in which genetic background, transgene expression levels and post-translational proteolytic processing can be controlled. These studies reveal that among the naturally occurring N-terminal HTT peptides, the exon 1 peptide is exceptionally pathogenic and exhibits unique structural and biophysical behaviors that do not appear to be incremental changes compared with other fragments. If this proves correct, efforts to specifically reduce the levels of exon 1 peptides or to target toxicity-influencing post-translational modifications that occur with the exon 1 context are likely to have the greatest impact on pathology.

Convergent replication of mouse synthetic prion strains.

Prion diseases are neurodegenerative disorders characterized by the aberrant folding of endogenous proteins into self-propagating pathogenic conformers. Prion disease can be initiated in animal models by inoculation with amyloid fibrils formed from bacterially derived recombinant prion protein. The synthetic prions that accumulated in infected organisms are structurally distinct from the amyloid preparations used to initiate their formation and change conformationally on repeated passage. To investigate the nature of synthetic prion transformation, we infected mice with a conformationally diverse set of amyloids and serially passaged the resulting prion strains. At each passage, we monitored changes in the biochemical and biological properties of the adapting strain. The physicochemical properties of each synthetic prion strain gradually changed on serial propagation until attaining a common adapted state with shared physicochemical characteristics. These results indicate that synthetic prions can assume multiple intermediate conformations before converging into one conformation optimized for in vivo propagation.

Conformational features of tau fibrils from Alzheimer's disease brain are faithfully propagated by unmodified recombinant protein.

Fibrils composed of tau protein are a pathological hallmark of several neurodegenerative disorders including Alzheimer's disease (AD). Here we show that when recombinant tau protein is seeded with paired helical filaments (PHFs) isolated from AD brain, the amyloid formed shares many of the structural features of AD PHFs. In contrast, tau amyloids formed with heparin as an inducing agent-a common biochemical model of tau misfolding-are structurally distinct from brain-derived PHFs. Using ultrastructural analysis by electron microscopy, circular dichroism, and chemical denaturation, we found that AD seeded recombinant tau fibrils were not significantly different than tau fibrils isolated from AD brain tissue. Tau fibrils produced by incubating recombinant tau with heparin had significantly narrower fibrils with a longer periodicity, higher chemical stability, and distinct secondary structure compared to AD PHFs. The addition of heparin to the reaction of recombinant tau and AD PHFs also corrupted the templating process, resulting in a mixture of fibril conformations. Our results suggest that AD-isolated PHFs act as a conformational template for the formation of recombinant tau fibrils. Therefore, the use of AD PHFs as seeds to stimulate recombinant tau amyloid formation produces synthetic tau fibers that closely resemble those associated with AD pathology and provides a biochemical model of tau misfolding that may be of improved utility for structural studies and drug screening. These results also demonstrate that post-translational modifications such as phosphorylation are not a prerequisite for the propagation of the tau fibril conformation found in AD.

Protein misfolding detected early in pathogenesis of transgenic mouse model of Huntington disease using amyloid seeding assay.

Huntington disease (HD) is one of several fatal neurodegenerative disorders associated with misfolded proteins. Here, we report a novel method for the sensitive detection of misfolded huntingtin (HTT) isolated from the brains of transgenic (Tg) mouse models of HD and humans with HD using an amyloid seeding assay (ASA), which is based on the propensity of misfolded proteins to act as a seed and shorten the nucleation-associated lag phase in the kinetics of amyloid formation in vitro. Using synthetic polyglutamine peptides as the substrate for amyloid formation, we found that partially purified misfolded HTT obtained from end-stage brain tissue of two Tg HD mouse models and brain tissue of post-mortem human HD patients was capable of specifically accelerating polyglutamine amyloid formation compared with unseeded reactions and controls. Alzheimer and prion disease brain tissues did not do so, demonstrating the specificity of the ASA. It is unclear whether early intermediates or later conformational species in the protein misfolding process act as seeds in the ASA for HD. However, we were able to detect misfolded protein in the brains of YAC128 mice early in disease pathogenesis (11 weeks of age), whereas large inclusion bodies have not been observed in the brains of these mice by histology until 78 weeks of age, much later in the pathogenic process. The sensitive detection of misfolded HTT protein early in the disease pathogenesis in the YAC128 Tg mouse model strengthens the argument for a causative role of protein misfolding in HD.

Protease-sensitive synthetic prions.

Prions arise when the cellular prion protein (PrP(C)) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc). Frequently, PrP(Sc) is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164), denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174) did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP(Sc) and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600-750 days in Tg4053 mice, which exhibited sPrP(Sc). These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP(Sc).

Design and construction of diverse mammalian prion strains.

Prions are infectious proteins that encipher biological information within their conformations; variations in these conformations dictate different prion strains. Toward elucidating the molecular language of prion protein (PrP) conformations, we produced an array of recombinant PrP amyloids with varying conformational stabilities. In mice, the most stable amyloids produced the most stable prion strains that exhibited the longest incubation times, whereas more labile amyloids generated less stable strains and shorter incubation times. The direct relationship between stability and incubation time of prion strains suggests that labile prions are more fit, in that they accumulate more rapidly and thus kill the host faster. Although incubation times can be changed by altering the PrP expression level, PrP sequence, prion dose, or route of inoculation, we report here the ability to modify the incubation time predictably in mice by modulating the prion conformation.

Natural and synthetic prion structure from X-ray fiber diffraction.

A conformational isoform of the mammalian prion protein (PrP(Sc)) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-beta diffraction: meridional intensity at 4.8 A resolution, indicating the presence of beta strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four beta-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brain-derived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 A meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 A, absent from brain-derived prions, and indicating the presence of stacked beta-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.

Small-molecule aggregates inhibit amyloid polymerization.

Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited beta-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus.

Prion detection by an amyloid seeding assay.

Polymerization of recombinant prion protein (recPrP), which was produced in bacteria, into amyloid fibers was accompanied by the acquisition of prion infectivity. We report here that partially purified preparations of prions seed the polymerization of recPrP into amyloid as detected by a fluorescence shift in the dye Thioflavin T. Our amyloid seeding assay (ASA) detected PrP(Sc), the sole component of the prion, in brain samples from humans with sporadic Creutzfeldt-Jakob disease, as well as in rodents with experimental prion disease. The ASA detected a variety of prion strains passaged in both mice and hamsters. The sensitivity of the ASA varied with strain type; for hamster Sc237 prions, the limit of detection was approximately 1 fg. Some prion strains consist largely of protease-sensitive PrP(Sc) (sPrP(Sc)), and these strains were readily detected by ASA. Our studies show that the ASA provides an alternative methodology for detecting both sPrP(Sc) and protease-resistant PrP(Sc) that does not rely on protease digestion or immunodetection.

Designing well-defined photopolymerized synthetic matrices for three-dimensional culture and differentiation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are of interest for the study of disease, where these cells can be derived from patients and have the potential to be differentiated into any cell type; however, three-dimensional (3D) culture and differentiation of iPSCs within well-defined synthetic matrices for these applications remains limited. Here, we aimed to establish synthetic cell-degradable hydrogels that allow precise presentation of specific biochemical cues for 3D culture of iPSCs with relevance for hypothesis testing and lineage-specific differentiation. We synthesized poly(ethylene glycol)-(PEG)-peptide-based hydrogels by photoinitiated step growth polymerization and used them to test the hypothesis that the viability of iPSCs within these matrices could be rescued with appropriate biochemical cues inspired by proteins and integrins important for iPSC culture on Matrigel. Specifically, we selected a range of motifs inspired by iPSC binding to Matrigel, including laminin-derived IKVAV and YIGSR, α5ß1-binding PHSRNG10RGDS, αvß5-binding KKQRFRHRNRKG, and RGDS that is known to bind a variety of integrins for generally promoting cell adhesion. YIGSR and PHSRNG10RGDS resulted in the highest iPSC viability, where binding of ß1 integrin was key, and these permissive compositions also allowed iPSC differentiation into neural progenitor cells (NPCs) (decreased oct4 expression and increased pax6 expression) in response to soluble factors. The resulting NPCs formed clusters of different sizes in response to each peptide, suggesting that matrix biochemical cues affect iPSC proliferation and clustering in 3D culture. In summary, we have established photopolymerizable synthetic matrices for the encapsulation, culture, and differentiation of iPSCs for studies of cell-matrix interactions and deployment in disease models.

Inert and seed-competent tau monomers suggest structural origins of aggregation.

Tauopathies feature progressive accumulation of tau amyloids. Pathology may begin when these amplify from a protein template, or seed, whose structure is unknown. We have purified and characterized distinct forms of tau monomer-inert (Mi) and seed-competent (Ms). Recombinant Ms triggered intracellular tau aggregation, induced tau fibrillization in vitro, and self-assembled. Ms from Alzheimer's disease also seeded aggregation and self-assembled in vitro to form seed-competent multimers. We used crosslinking with mass spectrometry to probe structural differences in Mi vs. Ms. Crosslinks informed models of local peptide structure within the repeat domain which suggest relative inaccessibility of residues that drive aggregation (VQIINK/VQIVYK) in Mi, and exposure in Ms. Limited proteolysis supported this idea. Although tau monomer has been considered to be natively unstructured, our findings belie this assumption and suggest that initiation of pathological aggregation could begin with conversion of tau monomer from an inert to a seed-competent form.

The N-terminus of the prion protein is a toxic effector regulated by the C-terminus.

PrPC, the cellular isoform of the prion protein, serves to transduce the neurotoxic effects of PrPSc, the infectious isoform, but how this occurs is mysterious. Here, using a combination of electrophysiological, cellular, and biophysical techniques, we show that the flexible, N-terminal domain of PrPC functions as a powerful toxicity-transducing effector whose activity is tightly regulated in cis by the globular C-terminal domain. Ligands binding to the N-terminal domain abolish the spontaneous ionic currents associated with neurotoxic mutants of PrP, and the isolated N-terminal domain induces currents when expressed in the absence of the C-terminal domain. Anti-PrP antibodies targeting epitopes in the C-terminal domain induce currents, and cause degeneration of dendrites on murine hippocampal neurons, effects that entirely dependent on the effector function of the N-terminus. NMR experiments demonstrate intramolecular docking between N- and C-terminal domains of PrPC, revealing a novel auto-inhibitory mechanism that regulates the functional activity of PrPC.

Development of a human light chain variable domain (V(L)) intracellular antibody specific for the amino terminus of huntingtin via yeast surface display.

Intracellular antibodies (intrabodies) provide an attractive means for manipulating intracellular protein function, both for research and potentially for therapy. A challenge in the isolation of effective intrabodies is the ability to find molecules that exhibit sufficient binding affinity and stability when expressed in the reducing environment of the cytoplasm. Here, we have used yeast surface display of proteins to isolate novel scFv clones against huntingtin from a non-immune human antibody library. We then applied yeast surface display to affinity mature this scFv pool and analyze the location of the binding site of the mutant with the highest affinity. Interestingly, the paratope was mapped exclusively to the variable light chain domain of the scFv. A single domain antibody was constructed consisting solely of this variable light chain domain, and was found to retain full binding activity to huntingtin. Cytoplasmic expression levels in yeast of the single domain were at least fivefold higher than the scFv. The ability of the single-domain intrabody to inhibit huntingtin aggregation, which has been implicated in the pathogenesis of Huntington's disease (HD), was confirmed in a cell-free in vitro assay as well as in a mammalian cell culture model of HD. Significantly, a single-domain intrabody that is functionally expressable in the cytoplasm was derived from a non-functional scFv by performing affinity maturation and binding site analysis on the yeast cell surface, despite the differences between the cytoplasmic and extracellular environment. This approach may find application in the development of intrabodies to a wide variety of intracellular targets.

Conformation-dependent epitopes recognized by prion protein antibodies probed using mutational scanning and deep sequencing.

Prion diseases are caused by a structural rearrangement of the cellular prion protein, PrP(C), into a disease-associated conformation, PrP(Sc), which may be distinguished from one another using conformation-specific antibodies. We used mutational scanning by cell-surface display to screen 1341 PrP single point mutants for attenuated interaction with four anti-PrP antibodies, including several with conformational specificity. Single-molecule real-time gene sequencing was used to quantify enrichment of mutants, returning 26,000 high-quality full-length reads for each screened population on average. Relative enrichment of mutants correlated to the magnitude of the change in binding affinity. Mutations that diminished binding of the antibody ICSM18 represented the core of contact residues in the published crystal structure of its complex. A similarly located binding site was identified for D18, comprising discontinuous residues in helix 1 of PrP, brought into close proximity to one another only when the alpha helix is intact. The specificity of these antibodies for the normal form of PrP likely arises from loss of this conformational feature after conversion to the disease-associated form. Intriguingly, 6H4 binding was found to depend on interaction with the same residues, among others, suggesting that its ability to recognize both forms of PrP depends on a structural rearrangement of the antigen. The application of mutational scanning and deep sequencing provides residue-level resolution of positions in the protein-protein interaction interface that are critical for binding, as well as a quantitative measure of the impact of mutations on binding affinity.

Prefibrillar huntingtin oligomers isolated from HD brain potently seed amyloid formation.

Many neurodegenerative diseases are associated with deposits of aggregated protein in the brain. The molecular pathways through which soluble proteins misfold to form amyloids and large protein aggregates often include diverse oligomeric species, only some of which progress to the amyloid state. Here we show that prefibrillar huntingtin (HTT) oligomers, isolated from Huntington's disease (HD) affected human brain samples or mouse models, stimulate polyglutamine amyloid formation. Fibrillar HTT oligomers have been shown to be unstable under denaturing conditions and appear not to lead to amyloid formation. Here we show that prefibrillar HTT oligomers are remarkably stable and are potent seeds of polyglutamine amyloid formation. Therefore, our findings help to dissect the complex molecular pathway of HTT misfolding.

Prions.

The discovery of infectious proteins, denoted prions, was unexpected. After much debate over the chemical basis of heredity, resolution of this issue began with the discovery that DNA, not protein, from pneumococcus was capable of genetically transforming bacteria (Avery et al. 1944). Four decades later, the discovery that a protein could mimic viral and bacterial pathogens with respect to the transmission of some nervous system diseases (Prusiner 1982) met with great resistance. Overwhelming evidence now shows that Creutzfeldt-Jakob disease (CJD) and related disorders are caused by prions. The prion diseases are characterized by neurodegeneration and lethality. In mammals, prions reproduce by recruiting the normal, cellular isoform of the prion protein (PrP(C)) and stimulating its conversion into the disease-causing isoform (PrP(Sc)). PrP(C) and PrP(Sc) have distinct conformations: PrP(C) is rich in α-helical content and has little ß-sheet structure, whereas PrP(Sc) has less α-helical content and is rich in ß-sheet structure (Pan et al. 1993). The conformational conversion of PrP(C) to PrP(Sc) is the fundamental event underlying prion diseases. In this article, we provide an introduction to prions and the diseases they cause.

De novo generation of prion strains.

Prions are self-replicating proteins that can cause neurodegenerative disorders such as bovine spongiform encephalopathy (also known as mad cow disease). Aberrant conformations of prion proteins accumulate in the central nervous system, causing spongiform changes in the brain and eventually death. Since the inception of the prion hypothesis - which states that misfolded proteins are the infectious agents that cause these diseases - researchers have sought to generate infectious proteins from defined components in the laboratory with varying degrees of success. Here, we discuss several recent studies that have produced an array of novel prion strains in vitro that exhibit increasingly high titres of infectivity. These advances promise unprecedented insight into the structure of prions and the mechanisms by which they originate and propagate.

A disulfide-free single-domain V(L) intrabody with blocking activity towards huntingtin reveals a novel mode of epitope recognition.

We present the crystal structure and biophysical characterization of a human V(L) [variable domain immunoglobulin (Ig) light chain] single-domain intrabody that binds to the huntingtin (Htt) protein and has been engineered for antigen recognition in the absence of its intradomain disulfide bond, otherwise conserved in the Ig fold. Analytical ultracentrifugation demonstrated that the αHtt-V(L) 12.3 domain is a stable monomer under physiological conditions even at concentrations >20 µM. Using peptide SPOT arrays, we identified the minimal binding epitope to be EKLMKAFESLKSFQ, comprising the N-terminal residues 5-18 of Htt and including the first residue of the poly-Gln stretch. X-ray structural analysis of αHtt-V(L) both as apo protein and in the presence of the epitope peptide revealed several interesting insights: first, the role of mutations acquired during the combinatorial selection process of the αHtt-V(L) 12.3 domain-initially starting from a single-chain Fv fragment-that are responsible for its stability as an individually soluble Ig domain, also lacking the disulfide bridge, and second, a previously unknown mode of antigen recognition, revealing a novel paratope. The Htt epitope peptide adopts a purely α-helical structure in the complex with αHtt-V(L) and is bound at the base of the complementarity-determining regions (CDRs) at the concave ß-sheet that normally gives rise to the interface between the V(L) domain and its paired V(H) (variable domain Ig heavy chain) domain, while only few interactions with CDR-L1 and CDR-L3 are formed. Notably, this noncanonical mode of antigen binding may occur more widely in the area of in vitro selected antibody fragments, including other Ig-like scaffolds, possibly even if a V(H) domain is present.

Stochastic kinetics of intracellular huntingtin aggregate formation.

Neurodegeneration in Huntington disease is described by neuronal loss in which the probability of cell death remains constant with time. However, the quantitative connection between the kinetics of cell death and the molecular mechanism initiating neurodegeneration remains unclear. One hypothesis is that nucleation of protein aggregates containing exon I fragments of the mutant huntingtin protein (mhttex1), which contains an expanded polyglutamine region in patients with the disease, is the explanation for the infrequent but steady occurrence of neuronal death, resulting in adult onset of the disease. Recent in vitro evidence suggests that sufficiently long polyglutamine peptides undergo a unimolecular conformational change to form a nucleus that seeds aggregation. Here we use this nucleation mechanism as the basis to derive a stochastic mathematical model describing the probability of aggregate formation in cells as a function of time and mhttex1 protein concentration, and validate the model experimentally. These findings suggest that therapeutic strategies for Huntington disease predicated on reducing the rate of mhttex1 aggregation need only make modest reductions in huntingtin expression level to substantially increase the delay time until aggregate formation.