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Reporters of Amyloid Structural Polymorphism
Chapter 7
Reporters of Amyloid Structural Polymorphism Harry LeVine III Department of Molecular and Cellular Biochemistry, Center on Aging, Center for Structural Biology, University of Kentucky, Lexington, KY, USA
K. Peter, R. Nilsson and Per Hammarström Department of Chemistry, IFM, Linköping University, Linköping, Sweden
Chapter Outline Background and Rationale 69 Ligand-Binding Phenotype 69 Conformational/Configurational Antibodies 70 Ligand-Binding Sites 70 Peptides70 Cerebrovascular Amyloid 70 Imaging Non-Aβ Amyloid Brain Pathology 70
BACKGROUND AND RATIONALE Misfolding of proteins induced by environmental conditions or by the presence of destabilizing mutations often triggers sequestration (aggresome formation) or cellular removal (unfolded protein response (UPR), autophagy) intracellu lar responses. Extracellular aggregates are phagocytosed or endocytosed by macrophages in the periphery and micro glia and astrocytes in the brain and central nervous system. In some cases a highly stable alternative assembly structure, an amyloid fibril, is formed that is highly resistant to deg radation and thus accumulates. The original definition of amyloid fibrils included only multimeric fibrillar structures whose fiber X-ray diffraction pattern revealed a high con tent of cross-β sheet secondary structural elements [1]. The definition has expanded in some systems to include assem blies containing a cross-β sheet secondary structure but not necessarily highly organized fibrillar morphology. Differ ent unrelated proteins can form amyloid fibrils, although a given fibril is a non-covalent structural polymer of only one protein and/or its fragments. The World Health Orga nization (WHO) (2012) recognizes 30 amyloid proteins and 8 intracellular inclusion bodies in humans classified by their secondary structure content, fibrillar morphology by Bio-nanoimaging. http://dx.doi.org/10.1016/B978-0-12-394431-3.00007-9 Copyright © 2014 Elsevier Inc. All rights reserved.
Probes that Report their Conformation and Environment 71 Hydrophobic Probes and Molecular Rotors 71 Luminescent Conjugated Oligo- and Polythiophenes 73 Assessing Pathology Spread and Quantification 75 Applications and Implications of Polymorphism-Sensitive Reporters77 Diagnostics77 Therapeutics77
electron microscopy, and birefringent staining with the azo cotton dye, Congo Red. These features represent common ality among amyloid fibrils and define the current classifi cation criteria for an amyloid. More recently recognized is the ability of amyloid fibrils to propagate or seed by addition of cognate protein mono mers to fibril ends by a process of conformational conver sion, resulting in fibril growth. Initially observed as part of the infectivity process of prions, this templating activity, analogous to crystal seeding in which the structural/confor mational properties of the seed are faithfully replicated, is another common property of amyloid fibrils that has conse quences for pathology as well as for applications as nano materials and their toxicology.
LIGAND-BINDING PHENOTYPE Before Congo Red was discovered to characteristically stain amyloid fibrils with linear birefringence, Nature had evolved serum amyloid P protein (SAP), a calcium-dependent pentameric protein of the pentraxin family, which rec ognizes the common amyloid fibril structural fold and occurs bound to extracellular amyloid fibrils, perhaps to shield amyloid fibrils from toxic interactions or to possibly
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assist in their removal. Imaging with radiolabeled SAP injected into patients [2] reveals the distribution of amy loid fibril deposits in peripheral organs and connective tis sue and can be used to assess the effectiveness of therapies designed to remove the deposits.
Conformational/Configurational Antibodies Recently, a number of antibodies have been produced that recognize amyloid fibrils in a primary sequence-indepen dent fashion [3,4]. Other antibodies recognize only nonfibrillar oligomeric forms of amyloidogenic proteins and peptides but not the monomeric forms [5–11]. While these antibodies are believed to recognize conformational fea tures that differentiate among the multimeric species, the molecular bases of the epitopes remain to be defined for most antibodies. Radiolabeled SAP and antibodies have proven useful in peripheral applications outside of the brain, but they have limited ability to reach the brain. Imaging the amyloid lesions (Aβ, tau, α-synuclein) involved in neurodegenera tive diseases which account for the vast majority of cases of amyloidoses requires high-affinity small molecules that will penetrate the blood–brain barrier and, if not bound, can be rapidly cleared from the brain. High affinity is required to result in sufficient quantities of radioligand binding to the pathologic lesions at the nanomolar ligand concentra tions attainable in vivo for reliable quantification. The first clinically useful brain-imaging ligand was the uncharged thioflavin T analog [11] C-labeled benzothiazole Pitts burgh Compound B (PIB) introduced in 2002, followed by the more user-friendly longer radioactive half life [18] F-labeled stilbene derivatives Florbetapir (AV-45) (Amyvid, approved in 2012) and Florbetaben (filing was anticipated in the 3rd quarter of 2012).
Ligand-Binding Sites Analysis of the in vitro binding of different ligand structures to synthetic Aβ fibrils by Lockhart et al [12,13] indicated that there were distinct binding sites for ligands that could accommodate different chemical structures, and that the density of the different sites were different [BS3 (Me-BTA1) << BS1 (thioflavin T) < BS2 (Congo Red)]. The detailed molecular basis for these binding sites is unknown because of the difficulties obtaining high-resolution fibril structures, and modeling studies remain to be validated by experimen tal evidence. Congo Red sites (BS2) were the most abun dant, 1:1 with Aβ molecules in the fibrils. The other ligand sites on Aβ fibrils were less abundant. A key observation was that, although Congo Red (BS2) binding to synthetic Aβ fibrils remained fairly constant (CR/Aβ monomer), the fibrils induced the characteristic fluorescence change in thioflavin T (BS1) with an efficiency dependent on the fibril
formation conditions which also modulate fibril morphol ogy [14]. This suggested that the ThT binding and/or fluo rescent properties were sensitive to fibril polymorphism, with Congo Red less so. The potential disease relevance of Aβ fibril polymorphism was highlighted by the observation that PIB (BS3) binding density was very low in synthetic Aβ fibrils, and in animal models of Aβ pathology (trans genic mice [15] and (LeVine unpublished), nonhuman pri mates [16], and canines (LeVine, unpublished), but high in humans with AD and Down syndrome [17] despite similar amounts of Aβ amyloid pathology. A hypothesis that can be drawn from these observations is that specific polymorphic forms of Aβ amyloid (parenchymal and vascular) fibrils are related to disease status because only humans progress to dementia.
Peptides The selective templating of Aβ peptides by synthetic Aβ fibrils and AD Aβ pathology has permitted proof-of-concept labeling of Aβ pathology with tagged Aβ peptides in animal models [18,19], but the challenges of brain penetration with peptides and their derivatives compared to small-molecule ligands have hindered widespread application of this tech nology.
Cerebrovascular Amyloid Cerebrovascular accumulation of Aβ(1-40) (Aβ1-40) in the smooth-muscle layer around blood vessels (amyloid angi opathy) is associated with vessel fragility and small-vessel hemorrhage [20]. Vascular amyloid is comorbid with Aβ(1-42)-containing parenchymal Aβ plaques in AD, and it binds Congo Red and its analogs as well as the thiofla vines. A recent report identifies certain resorufin analogs as selective for cerebrovascular amyloid angiopathy over parenchymal Aβ deposits, occupying a distinct binding site. Current cerebrovascular-specific amyloid ligands lack sufficient affinity for in vivo imaging [21]. The molecular basis for this selectivity and its relationship to the BS(13) ligand binding sites on synthetic and parenchymal Aβ fibrils remains to be determined.
Imaging Non-Aβ Amyloid Brain Pathology Specific ligands that could distinguish individual amyloid proteins by in vivo imaging have been elusive. Consistent with general Congo Red binding to the defining cross-β sheet structure in amyloid fibrils, the BS2 site seems to be ubiquitously present on amyloid fibrils formed from a vari ety of proteins. The BS2 site is also present on neurofibril lary tau tangles which bind Congo Red and thioflavine S, commensurate with their amyloid character, but the affin ity and amount of tau lesions for PIB (BS3) are too low for imaging. Tau-selective ligands have been identified;
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however, they lack the requisite affinity and sufficient selectivity to distinguish tau from the more abundant Aβ in AD and in other mixed pathologies [22–25]. Similarly, α-synuclein cannot be imaged with PIB in humans [26]. Thus, high-affinity PIB binding is specific for Aβ fibrillar pathology of neurodegenerative diseases. Recently, Taghavi and co-workers [25] showed that the structure–activity relationship of N-benzylidene-benzohy drazide (NBB) binding to tau and Amyloid(1-42) fibrils indicated differential selectivity for these protein aggre gates. The NBB, BSc4014, demonstrated selective tangle staining, whereas NBB BSc3869 exhibits selective Aβ plaque staining.
PROBES THAT REPORT THEIR CONFORMATION AND ENVIRONMENT The most common small amyloid ligands are derivatives of Congo Red or the benzothiazole thioflavins. These probes are rather selective for protein aggregates having an exten sive content of cross β-pleated sheet conformation and structural regularity. However, it is evident that significant morphologic variation can exist between different amy loid fibrils formed from the same peptide or protein. Most conventional amyloid probes, with the exception of certain antibodies, do not distinguish protein aggregates of diverse morphologic origin at the light microscopic level. Recent evidence indicates that pre-fibrillar states preceding the for mation of well-defined amyloid fibrils are likely to play a critical role in the pathogenesis of protein aggregation dis eases [27–29]. The small-molecule-based amyloid ligands fail to recognize these pre-fibrillar species. Hence, there is a need for small-molecule amyloid ligands that identify a broader subset of proteinaceous aggregated species and variety of morphologically distinct protein aggregates.
Hydrophobic Probes and Molecular Rotors Many amyloid probe targeting studies have employed fluorescence spectroscopy to take advantage of the intrin sic sensitivity, applicability to histology, and potential for reporting the molecular environment of the probe. Several of the molecular scaffolds for in vitro or ex vivo detection have been developed for in vivo radioisotope PET or SPECT imaging of amyloid in the clinic (naphthalenes (FDDNP), styrylbenzenes (florbetapir, florbetaben), benzothiazoles (PIB)) (Fig. 7.2B). Amyloid probes are frequently based on substituted aro matic ring systems, such as benzophenoxazine, benzothiazole, benzoxazole, benzofuran, imidazopyridine, napthalene, stil bene, thiophene, or combinations thereof. Molecular probes with an extended linear configuration have been most widely studied. Their selectivity and affinity for amyloid are likely due to binding in deep elongated grooves in the amyloid fibril
FIGURE 7.1 High-resolution model of Congo Red bound to an amyloid fibril composed of the 218–289 fragment of HET-s. The fibril structure is rendered in light green and the Congo Red molecule as a space-filling model. The planarized Congo Red molecule binds in a hydrophobic groove aligned along the fibril axis. Specific alignment of the binding site was dictated by ionic bonds between the sulfonate side groups of Congo Red and the residue Lys229 in the amyloid fibril. The figure was made in PyMol using the pdb code 2LBU.
surface perpendicular to the fibril axis defined by the crossbeta sheet packing of the secondary structure as well as the zipper-like configuration of the constituent side-chains form ing the groove wall (Fig. 7.1). A high-resolution model of such a binding mode was recently presented for Congo Red bound to the yeast prion protein HET-s [30] which is suggested to be the BS2-type of binding site common to many different amy loid fibrils. The preference for linear probes binding to mature fibrils, rather than to early formed prefibrillar oligomers, is suggested by the fibril binding of ThT and 1-anilinonaphtha lene-8-sulfonate (ANS), while the fluorescence of the bulkier 4-(dicyanovinyl)-julolidine (DCVJ) and 4, 4-bis-1-phenylamino-8-naphthalene sulfonate (bis-ANS) increased in the presence of the prefibrillar oligomers [31] This observation suggests that selective prefibrillar probes should be less planar and less linear or possess a flexible backbone (see LCO sec tion below) to accommodate such configurations. The physicochemical basis for the increased fluores cence of molecular probes upon binding to prefibrillar states and to amyloid fibrils can be attributed mainly to two effects: (1) intrinsic molecular quenching, and (2) sol vent quenching and polarization. Molecular rotors (DDNP, DCVJ and NIAD-4, ANCA) (Fig. 7.2) are intrinsically
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FIGURE 7.2 (A) Chemical structures of small hydrophobic probes and molecular rotors. (B) Chemical structures of amyloid ligands.
Chapter | 7 Polymorphism Reporters
quenched in solution due to rapid rotation of quenching sub stituents. Steric restriction upon binding to an oligomeric state or to an amyloid fibril reduces the quenching. The amyloid fibril-induced fluorescence of ThT is suggested to occur by this mechanism [31]. Solvent quenching or polar ization of excited-state probes which are readily polarizable (CRANAD-2, ANS, NIAD-4, bis-ANS, methoxy-X04, X-34, and Nile Red) (Fig. 7.2) are stabilized by water but are much less affected by a hydrophobic milieu. A small Stokes shift with an enhanced fluorescence quantum yield is observed in hydrophobic environments. An example of a probe reporting molecular character istics of the ligand-binding sites on amyloid fibrils is the benzophenoxazine-based dye Nile Red, which can eluci date the local polarity of its fibril binding site in solution for several amyloidogenic proteins (Aβ, insulin, lysozyme, prion protein, and transthyretin [32]). The dielectric con stant for the binding pocket was estimated by calibrating the fluorescence of unbound probe in a series of organic solvents. Some of the amyloid fibril binding sites for Nile Red were more hydrophobic than octanol, a solvent mod eling the dielectric of the hydrophobic core of globular proteins. The structural similarity between Nile Red and extended amyloid probes, as well as partial competition with ThT [32], suggests that its binding site is BS2 and/or BS1. Nile Red does not change fluorescence in the pres ence of oligomeric prefibrillar states, instead targeting more mature amyloid fibrils. Unfortunately, Nile Red is too lipophilic for specific binding to amyloid in complex tissue environments, a property exploited in its use as a histologi cal lipid probe [33]. Aminonaphthlanene 2-cyanoacrylate (ANCA) is an example of a class of probes that, like Nile Red, can dis tinguish differences in amyloid binding-site polarity, but it appears to be selective enough for amyloid imaging in tissue sections [34]. The ANCA class of probes displays an amy loid fibril-induced molecular rotor fluorescence response (intrinsic quenching and polarizability). Aggregated states of proteins which have not attained the final mature form of an amyloid fibril are large com pared to soluble globular proteins or unfolded peptides and they display exposed hydrophobic patches, characteristics addressable for selective probe targeting. Bulky and flexible molecules are more likely than linear structures to detect these species. ANS has been reported to detect cytotoxic oligomeric states in vitro [35]. The authors suggest that the amount of exposed organized hydrophobic surface of the oligomers quantified by ANS binding is correlated with cytotoxicity due to disturbance of the membrane bilayer or membrane proteins. Probes that can directly delineate surface characteristics of amyloidogenic states in vivo will advance future research, and will be applicable to diagnos tics and therapeutics.
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Luminescent Conjugated Oligo- and Polythiophenes Luminescent conjugated polythiophenes (LCPs) with molecular scaffolds consisting of polymers of thiophene moieties have evolved as an interesting and uniquely use ful class of fluorescent probes. These molecules provide a distinctive optical read-out through the impact of detailed biomolecular interactions on the conformation and the geometry of the LCP backbone [36–39]. The structurally induced optical changes of the LCP backbone also allow the tantalizing prospect of detection of altered conforma tions of biomolecules. A variety of LCP-based amyloidspecific ligands have been reported (Fig. 7.3A) [40–42]. These ligands provide detailed morphology of the protein deposits and conformational phenotyping of distinct and polymorphic protein aggregates. Beyond reporting the total amount, the LCPs allow spectral fingerprinting of polymor phic populations of these assemblies. These ligands have proved effective for studying protein aggregates ex vivo and in vivo in transgenic mouse models with AD Aβ pathol ogy [42,43], in transgenic mice infected with distinct prion strains [44–48], and in human clinical tissue samples [49]. Application of LCPs to transgenic mouse models with AD Aβ pathology revealed a striking heterogeneity in the characteristic plaques composed of the Aβ peptide [42]. LCP staining of brain tissue slices revealed distinct subpopulations of plaques, seen as plaques fluorescing different colors, and intraplaque heterogeneity of color. The spectral features of LCPs are useful for comparison of polymorphic protein aggregates in well-defined experimental systems. Polymorphic protein aggregates are found in many protein aggregation disorders, and this is especially notable in the infectious prion diseases. Prions can occur as differ ent strains that are most likely encoded in the tertiary or quaternary structure of the prion aggregates. LCP staining of protein aggregates in brain sections from mice infected with distinct prion strains exhibited a distinct spectroscopic signature when bound to strain-specific prion deposits [44]. Different prion strains could also be distinguished by their staining selectivity by LCPs with distinct ionic side chains. The spectral differences due to different thiophene back bone conformations for the anionic LCP, polythiophene acetic acid (PTAA), were visualized by correlation plots of the ratios of the intensities of fluorescence emission at specific wavelengths. Prion aggregates associated with distinct prion strains, chronic wasting disease (CWD), and sheep scrapie, were easily distinguished (Fig. 7.3B). LCP fluorescence analysis has also been used to identify novel prion strains [45] and to investigate the molecular basis for interspecies prion disease transmission in mice [46,47]. PTAA allows ready characterization and identification of mixed prion strains within a single host (Fig. 7.3C) [48].
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FIGURE 7.3 (A) Chemical structures of luminescent conjugated polythiophenes that have been utilized as amyloid ligands. (B) Fluorescence images and spectra (right) of prion deposits, chronic wasting disease (mCWD) (left) and sheep scrapie (mNS) (middle) stained by PTAA. Some representative PTAA-stained prion deposits are indicated by white arrows. (C) Fluorescence images of PTAA stained mCWD and mNS deposits in a single host. mCWD deposits have a green spectrum (middle), whereas mNS deposits have a red spectrum (right). (D) Fluorescence image of PTAA bound to amyloid deposits in tissue samples, heart (left) and liver (right) from a patient diagnosed with AA-amyloidosis. Two different colors are observed from distinct deposits, suggesting the presence of polymorphic AA-amyloid.
The presence of multiple prion strains is difficult to demon strate by conventional biochemical methods. Studies of prion deposits in tissue samples [44–48] sug gest that the LCP emission profile is an indirect readout of structural difference between prion deposits associated with
distinct prion strains. To mechanistically relate geometri cal alterations of the LCPs to structural variations of the protein deposits, rather than their composition, studies were carried out with recombinant prion protein, PrP, [44,50,51]. Different forms of in vitro amyloid fibrils generated from
Chapter | 7 Polymorphism Reporters
recombinant mouse, Syrian hamster or human prion pro tein (mPrP, sHaPrP, HuPrP) displayed distinct LCP spec tra. The emission profile of PTAA distinguished individual aggregates within different PrP fibril preparations that were chemically identical (same PrP sequence). Thus, spectral differences in tissue observed for PTAA were most likely due to structural differences between the fibrils. LCP fin gerprinting will be useful for the analysis of conformational polymorphism in other protein aggregation diseases, as similar phenomena may be much more frequent in other neurodegenerative protein aggregation disorders and amy loidoses [52]. PTAA emission spectral profiles can be used to sub-type systemic amyloidoses in tissue samples similar to prion strains [44]; PTAA staining revealed the existence of multiple types of AA amyloid in a single host (Fig. 7.3D) [49]. Although LCPs are uniquely suited to spectroscopic characterization of polymorphic amyloid deposits, they do not detect pre-fibrillar aggregates preceding the forma tion of mature amyloid fibrils. They are also relatively high molecular weight, polydisperse, and do not penetrate the blood–brain barrier for in vivo imaging of protein aggre gates in the brain. In 2009, Åslund and co-workers [53] introduced a novel class of smaller chemically defined LCPs, denoted luminescent conjugated oligothiophenes (LCOs), based on a pentameric thiophene backbone (Fig. 7.4A). These molecules are amyloid-specific ligands under physiologic conditions and show striking enhanced fluo rescence and distinct emission wavelength profiles when bound to protein aggregates associated with AD and prion diseases. The LCOs display a distinct emission profile with well-resolved sub-structure upon binding to recombinant Aβ(1-42) amyloid fibrils, implying that the backbone of the LCOs becomes more rigid upon binding to the fibrils (Fig. 7.4B). Both thioflavin T (ThT) and one of the LCOs, p-FTAA, reported conventional nucleated fibrillation kinetic behavior of recombinant Aβ(1-42) peptide, including a lag phase, a rapid exponential growth phase, and a final plateau phase. However, with the recombinant Aβ(1-40) peptide, pFTAA fluorescence revealed an earlier growth phase than ThT, indicating that p-FTAA detected pre-fibrillar Aβ(140) species preceding the formation of amyloid fibrils (Fig. 7.4B). p-FTAA also detects non-thioflavinophilic pre-fibril lar aggregates in vitro for a variety of other amyloidogenic proteins, including PrP, insulin, lysozyme and different Aβ peptides [54]. Using a small library of thiophenes with distinct chain length, Nilsson and co-workers were able to show that a thiophene backbone consisting of at least five thiophene units was necessary to detect the pre-fibrillar aggregated species [55]. The high selectivity for protein aggregates and the distinctive conformation-induced optical properties of the novel chemically defined LCOs were further demon strated when applied to cryo-sectioned brain tissue from
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AD patients (Fig. 7.4C). The major pathologic hallmarks of AD, Aβ deposits, neurofibrillary tangles (NFTs) and dystrophic neurites, were clearly detected by all of the LCOs. Moreover, the LCOs showed complete co-localiza tion with 6E10 and AT8, antibodies conventionally used to stain Aβ and phosphorylated tau in NFTs, respectively [53,55]. Pentameric, hexameric, and heptameric LCOs with the terminal thiophenes substituted with carboxyl groups at the α-position (Fig. 7.4A) gave different emis sion spectra when bound to the Aβ and tau pathologies in the human AD brain. This novel class of oligothiophenebased amyloid-specific dyes is a promising histologic tool for spectral assignment of distinct protein aggregates observed in AD. Surprisingly, anionic LCOs with four negative charges can be used for in vivo optical imaging of protein aggregates (Fig. 7.4D) [53]. The labeling of plaques in the transgenic APP/PS1 mouse brain can be observed in real time by mul tiphoton microscopy through a cranial window overlying the parietal cortex. The staining was persistent as individ ual LCO stained amyloid deposits were detectable even 1 week post-injection of the dye. p-FTAA also specifically labels prion deposits associated with distinct prion strains in vivo, and the strains could be distinguished by their p-FTAA spectral signature [55]. Lately, it was also shown that the heptameric LCO, h-FTAA, could be utilized for spectral assignment of CβAA, Aβ plaques and intracellular tau aggregates in transgenic mice after a single intravenous injection of the LCO [56]. Although the conformation-dependent spectral informa tion would be lost, radiolabeled versions of the oligomeric thiophenes for clinical PET imaging could be developed. Optimization of the thiophene core structure will be required to provide the requisite binding selectivities for the different protein pathologies, the different species of protein aggre gates (oligomers, fibrils), and their polymorphic forms.
ASSESSING PATHOLOGY SPREAD AND QUANTIFICATION The dissemination of misfolded protein pathology and neu ronal dysfunction of the major neurodegenerative diseases follows stereotypical patterns through the brain. While the specific brain regions involved differ between diseases, in keeping with their clinical presentation the spreading is reminiscent of the neuronal connectivity between the affected regions. The proposed mechanism is the transport of fragments or nuclei of misfolded Aβ, tau, or α-synuclein pathology within neurons which template fibril or soluble assembly growth from cognate monomeric species. Exocy tosis of misfolded seeds in exosomes and their uptake by other neurons has also been observed [57,58] and may con stitute another mechanism that could also account for the spread of pathology to non-connected brain regions.
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FIGURE 7.4 (A) Chemical structures of tetrameric (q-FTAA), pentameric (p-FTAA) and heptameric (h-FTAA) luminescent conjugated oligothiophenes that have been utilized as amyloid ligands. (B) Fluorescence spectrum (left) of p-FTAA mixed with recombinant Aβ(1-40) amyloid fibrils (green) or freshly dissolved Aβ(1-40) (blue). Comparison between ThT and p-FTAA for monitoring the kinetics of recombinant Aβ(1-40) amyloid fibril formation. Notably, p-FTAA reacts earlier than ThT. (C) Fluorescence image (left) and spectra (right) of p-FTAA bound to the two pathologic hallmarks, Aβ deposits (green) and neurofibrillary tangles (yellow-red), in brain tissue samples from an AD patient. (D) Fluorescence images after a single intravenous injection of hFTAA in transgenic mice. Characteristic amyloid lesions, cerebrovascular β-amyloid angiopathy (CβAA), Aβ plaques, and intracellular tau aggregates are labeled throughout the brain and visualized by green fluorescence from h-FTAA.
The fidelity of template structural features is frequently very high, restricting seeding mainly to the cognate peptide, although this can be altered in vitro, and there is some evi dence for direct or indirect heterologous templating between
pathologies [59]. The templating fidelity extends to polymor phic structural variants which are observable in the form of prion strains with different characteristics which can co-exist in the brains of infected animals but replicate independently
Chapter | 7 Polymorphism Reporters
[48,60]. This property is exploited to amplify specific confor mations of prions in analytical methods such as protein mis folding cyclic amplification (PMCA) [61] or to template the formation of sufficient quantities of a particular fibril poly morph for structural analysis from a biologic sample from a synthetic or recombinant monomer [62]. The persistence of in vivo pFTAA staining of amyloid deposits could be utilized to trace dispersal in animal models in which spectrally dis tinct LCOs could be applied as amyloid ‘time stamps’.
APPLICATIONS AND IMPLICATIONS OF POLYMORPHISM-SENSITIVE REPORTERS Diagnostics It is clear from the increasing scope and evolving polymor phic complexity of misfolded protein proteopathies that the original tools for diagnosis, Congo Red staining and bire fringence and silver stains for Aβ plaques and tau, cannot provide the depth and wealth of information that may be required for accurate early detection of the disease process and for probing the underlying biology. There remains a dearth of ligands specific for soluble oligomeric species of misfolded proteins. All pathologic lesions of the same type are not equal in their impact on, or reflection of, the disease. Newer probes, such as the Aβ fibril binding site subtype-specific ligands and the poly- and oligothiophene fluorophores, are beginning to fill this gap. Multiplexed mixtures of probes, or at least multiple determinations, may be required. The true impact of these new probes remains to be established as they are in an early stage of development and require further validation at the pathologic level.
Therapeutics Certainly by their diagnostic impact, the new probes can assist in determining whether particular therapeutic strate gies have had the anticipated effect on specific polymorphic forms of protein aggregates. Whether by their direct interac tion with misfolded protein pathologies similar molecules could induce the dissociation or detoxification of polymor phic bioactive species awaits to be conclusively determined. There have been reports that affording amyloid targeting, e.g. with the polyphenolic compound curcumin, which is structurally similar to the amyloid reporters described in this chapter, increases the rate of conversion from the putatively neurotoxic prefibrillar oligomer assemblies into mature amyloid fibrils, thereby reducing neurotoxicity in transgenic Drosophila expressing Aβ peptides [63]. A similar mechanism was reported for the curcumin-related Orcein class of small molecules, suggesting a commonal ity [64]. Testing a library of LCP molecules as therapeu tic agents against prions in organotypic cultured cerebellar slices revealed a dramatic structure-dependent differential
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reduction of infectivity while accumulated protease- resistant PrP increased [65]. This showed that LCP-induced hyperstabilization of PrP amyloid fibrils was an operational trapping mechanism for reducing infectivity by suppressing fragmentation. Selectively targeting specific polymorphic forms of amyloid may have potential therapeutic benefit for neurotoxic amyloidogenic proteins such as Aβ and PrP.
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[16] Rosen RF, Walker LC, LeVine 3rd H. PIB binding in aged primate brain: Enrichment of high-affinity sites in humans with Alzheimer’s disease. Neurobiol Aging 2011;32:223–34. [17] Landt J, D’Abrera JC, Holland AJ, Aigbirhio FI, Fryer TD, Canales R, et al. Using positron emission tomography and carbon 11-labeled Pittsburgh compound B to image brain fibrillar {beta}-amyloid in adults with Down syndrome: Safety, acceptability, and feasibility. Arch Neurol 2011;68:890–6. [18] Sumbria RK, Boado RJ, Pardridge WM. Imaging amyloid alaque in Alzheimer’s disease brain with a biotinylated abeta peptide radio pharmaceutical conjugated to an IgG-avidin fusion protein. Biocon jug Chem 2012;23:1318–21. [19] Kandimalla KK, Wengenack TM, Curran GL, Gilles EJ, Poduslo JF. Pharmacokinetics and amyloid plaque targeting ability of a novel peptide-based magnetic resonance contrast agent in wild type and Alzheimer’s disease transgenic mice. J Pharmacol Exp Ther 2007;318:17–25. [20] Samarasekera N, Smith C, Al-Shahi Salman R. The association between cerebral amyloid angiopathy and intracerebral haemor rhage: systematic review and meta-analysis. J Neurol Neurosurg Psychiatry 2012;83:275–81. [21] Han BH, Zhou ML, Vellimana AK, Milner E, Kim DH, Greenberg JK, et al. Resorufin analogs preferentially bind cerebrovascular amy loid: potential use as imaging ligands for cerebral amyloid angiopa thy. Mol Neurodegener 2011;6:86. [22] Zhang W, Arteaga J, Cashion DK, Chen G, Gangadharmath U, Gomez LF, et al. Highly selective and specific PET tracer for imag ing of tau pathologies. J Alzheimers Dis 2012;31:601–12. [23] Bolander A, Kieser D, Voss C, Bauer S, Schon C, Burgold S, et al. Bis(arylvinyl)pyrazines, -pyrimidines, and -pyridazines as imaging agents for tau fibrils and beta-amyloid plaques in Alzheimer’s disease models. J Med Chem 2012;55:9170–80. [24] Cisek K, Jensen JR, Honson NS, Schafer KN, Cooper GL, Kuret J. Ligand electronic properties modulate tau filament binding site den sity. Biophys Chem 2012;170:25–33. [25] Taghavi A, Nasir S, Pickhardt M, Heyny-von Haussen R, Mall G, Mandelkow E, et al. N’-benzylidene-benzohydrazides as novel and selective tau-PHF ligands. J Alzheimers Dis 2011;27: 835–43. [26] Mathis CA, Mason NS, Lopresti BJ, Klunk WE. Development of positron emission tomography beta-amyloid plaque imaging agents. Semin Nucl Med 2012;42:423–32. [27] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s dis ease: progress and problems on the road to therapeutics. Science 2002;297:353–6. [28] Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, et al. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 2002;416:535–9. [29] Tomic JL, Pensalfini A, Head E, Glabe CG. Soluble fibrillar oligomer levels are elevated in Alzheimer’s disease brain and correlate with cognitive dysfunction. Neurobiol Dis 2009;35:352–8. [30] Schutz AK, Soragni A, Hornemann S, Aguzzi A, Ernst M, Bock mann A, et al. The amyloid-Congo red interface at atomic resolution. Angew Chem Int Ed Engl 2011;50:5956–60. [31] Lindgren M, Sorgjerd K, Hammarstrom P. Detection and char acterization of aggregates, prefibrillar amyloidogenic oligo mers, and protofibrils using fluorescence spectroscopy. Biophys J 2005;88:4200–12.
[32] Mishra R, Sjolander D, Hammarstrom P. Spectroscopic characteriza tion of diverse amyloid fibrils in vitro by the fluorescent dye Nile red. Mol Biosyst 2011;7:1232–40. [33] Fowler SD, Greenspan P. Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O. J Histochem Cytochem 1985;33:833–6. [34] Cao K, Farahi M, Dakanali M, Chang WM, Sigurdson CJ, Theodora kis EA, et al. Aminonaphthalene 2-cyanoacrylate (ANCA) probes fluorescently discriminate between amyloid-beta and prion plaques in brain. J Am Chem Soc 2012;134:17338–41. [35] Bolognesi B, Kumita JR, Barros TP, Esbjorner EK, Luheshi LM, Crowther DC, et al. ANS binding reveals common features of cyto toxic amyloid species. ACS Chem Biol 2010;5:735–40. [36] Ho HA, Boissinot M, Bergeron MG, Corbeil G, Dore K, Boudreau D, et al. Colorimetric and fluorometric detection of nucleic acids using cationic polythiophene derivatives. Angew Chem Int Ed Engl 2002;41:1548–51. [37] Nilsson KP, Inganas O. Chip and solution detection of DNA hybrid ization using a luminescent zwitterionic polythiophene derivative. Nat Mater 2003;2:419–24. [38] Nilsson KPR, Inganäs O. Optical emission of a conjugated polyelec trolyte report calcium induced conformational changes in calmod ulin and calmodulin-calcineurin interactions. Macromolecules 2004;37:9109–13. [39] Nilsson KP, Rydberg J, Baltzer L, Inganas O. Self-assembly of synthetic peptides control conformation and optical properties of a zwitterionic polythiophene derivative. Proc Natl Acad Sci USA 2003;100:10170–4. [40] Nilsson KP, Hammarstrom P, Ahlgren F, Herland A, Schnell EA, Lindgren M, et al. Conjugated polyelectrolytes-conformation-sen sitive optical probes for staining and characterization of amyloid deposits. Chembiochem 2006;7:1096–104. [41] Aslund A, Herland A, Hammarstrom P, Nilsson KP, Jonsson BH, Inganas O, et al. Studies of luminescent conjugated polythiophene derivatives: enhanced spectral discrimination of protein conforma tional states. Bioconjug Chem 2007;18:1860–8. [42] Nilsson KP, Aslund A, Berg I, Nystrom S, Konradsson P, Herland A, et al. Imaging distinct conformational states of amyloid-beta fibrils in Alzheimer’s disease using novel luminescent probes. ACS Chem Biol 2007;2:553–60. [43] Philipson O, Hammarstrom P, Nilsson KP, Portelius E, Olofsson T, Ingelsson M, et al. A highly insoluble state of Abeta similar to that of Alzheimer’s disease brain is found in Arctic APP transgenic mice. Neurobiol Aging 2009;30:1393–405. [44] Sigurdson CJ, Nilsson KP, Hornemann S, Manco G, Polymenidou M, Schwarz P, et al. Prion strain discrimination using luminescent conjugated polymers. Nat Methods 2007;4:1023–30. [45] Sigurdson CJ, Nilsson KP, Hornemann S, Heikenwalder M, Manco G, Schwarz P, et al. De novo generation of a transmissible spon giform encephalopathy by mouse transgenesis. Proc Natl Acad Sci USA 2009;106:304–9. [46] Sigurdson CJ, Nilsson KP, Hornemann S, Manco G, FernandezBorges N, Schwarz P, et al. A molecular switch controls interspecies prion disease transmission in mice. J Clin Invest 2010;120:2590–9. [47] Sigurdson CJ, Joshi-Barr S, Bett C, Winson O, Manco G, Schwarz P, et al. Spongiform encephalopathy in transgenic mice expressing a point mutation in the beta2-alpha2 loop of the prion protein. J Neuro sci 2011;31:13840–7.
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[48] Nilsson KP, Joshi-Barr S, Winson O, Sigurdson CJ. Prion strain interactions are highly selective. J Neurosci 2010;30:12094–102. [49] Nilsson KP, Ikenberg K, Aslund A, Fransson S, Konradsson P, Rocken C, et al. Structural typing of systemic amyloidoses by luminescentconjugated polymer spectroscopy. Am J Pathol 2010;176:563–74. [50] Almstedt K, Nystrom S, Nilsson KP, Hammarstrom P. Amyloid fibrils of human prion protein are spun and woven from morphologi cally disordered aggregates. Prion 2009;3:224–35. [51] Ostapchenko VG, Sawaya MR, Makarava N, Savtchenko R, Nils son KP, Eisenberg D, et al. Two amyloid states of the prion pro tein display significantly different folding patterns. J Mol Biol 2010;400:908–21. [52] Aguzzi A. Cell biology: Beyond the prion principle. Nature 2009;459:924–5. [53] Aslund A, Sigurdson CJ, Klingstedt T, Grathwohl S, Bolmont T, Dickstein DL, et al. Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggre gates in cerebral amyloidoses. ACS Chem Biol 2009;4:673–84. [54] Hammarstrom P, Simon R, Nystrom S, Konradsson P, Aslund A, Nils son KP. A fluorescent pentameric thiophene derivative detects in vitroformed prefibrillar protein aggregates. Biochemistry 2010;49:6838–45. [55] Klingstedt T, Aslund A, Simon RA, Johansson LB, Mason JJ, Nys trom S, et al. Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates. Org Biomol Chem 2011;9:8356–70. [56] Wegenast-Braun BM, Skodras A, Bayraktar G, Mahler J, Fritschi SK, Klingstedt T, et al. Spectral discrimination of cerebral amyloid lesions after peripheral application of luminescent conjugated oligo thiophenes. Am J Pathol 2012;181(6):1953–60.
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[57] Vingtdeux V, Sergeant N, Buee L. Potential contribution of exosomes to the prion-like propagation of lesions in Alzheimer’s disease. Front Physiol 2012;3:229. [58] Saman S, Kim W, Raya M, Visnick Y, Miro S, Jackson B, et al. Exosome-associated tau is secreted in tauopathy models and is selec tively phosphorylated in cerebrospinal fluid (CSF) in early Alzheim er’s disease. J Biol Chem 2012;287:3842–9. [59] Jucker M, Walker LC. Pathogenic protein seeding in Alzheimer disease and other neurodegenerative disorders. Ann Neurol 2011;70:532–40. [60] Makarava N, Baskakov IV. The same primary structure of the prion protein yields two distinct self-propagating states. J Biol Chem 2008;283:15988–96. [61] Soto C, Saborio GP, Anderes L. Cyclic amplification of protein mis folding: application to prion-related disorders and beyond. Trends Neurosci 2002;25:390–4. [62] Piro JR, Wang F, Walsh DJ, Rees JR, Ma J, Supattapone S. Seeding specificity and ultrastructural characteristics of infectious recombi nant prions. Biochemistry 2011;50:7111–6. [63] Caesar I, Jonson M, Nilsson KP, Thor S, Hammarstrom P. Curcumin promotes A-beta fibrillation and reduces neurotoxicity in transgenic Drosophila. PLoS ONE 2012;7:e31424. [64] Bieschke J, Herbst M, Wiglenda T, Friedrich RP, Boeddrich A, Schiele F, et al. Small-molecule conversion of toxic oligomers to nontoxic beta-sheet-rich amyloid fibrils. Nat Chem Biol 2011;8: 93–101. [65] Margalith I, Suter C, Ballmer B, Schwarz P, Tiberi C, Sonati T, et al. Polythiophenes inhibit prion propagation by stabilizing prion protein (PrP) aggregates. J Biol Chem 2012;287:18872–87.
Reporters of Amyloid Structural Polymorphism Harry LeVine III Department of Molecular and Cellular Biochemistry, Center on Aging, Center for Structural Biology, University of Kentucky, Lexington, KY, USA
K. Peter, R. Nilsson and Per Hammarström Department of Chemistry, IFM, Linköping University, Linköping, Sweden
Chapter Outline Background and Rationale 69 Ligand-Binding Phenotype 69 Conformational/Configurational Antibodies 70 Ligand-Binding Sites 70 Peptides70 Cerebrovascular Amyloid 70 Imaging Non-Aβ Amyloid Brain Pathology 70
BACKGROUND AND RATIONALE Misfolding of proteins induced by environmental conditions or by the presence of destabilizing mutations often triggers sequestration (aggresome formation) or cellular removal (unfolded protein response (UPR), autophagy) intracellu lar responses. Extracellular aggregates are phagocytosed or endocytosed by macrophages in the periphery and micro glia and astrocytes in the brain and central nervous system. In some cases a highly stable alternative assembly structure, an amyloid fibril, is formed that is highly resistant to deg radation and thus accumulates. The original definition of amyloid fibrils included only multimeric fibrillar structures whose fiber X-ray diffraction pattern revealed a high con tent of cross-β sheet secondary structural elements [1]. The definition has expanded in some systems to include assem blies containing a cross-β sheet secondary structure but not necessarily highly organized fibrillar morphology. Differ ent unrelated proteins can form amyloid fibrils, although a given fibril is a non-covalent structural polymer of only one protein and/or its fragments. The World Health Orga nization (WHO) (2012) recognizes 30 amyloid proteins and 8 intracellular inclusion bodies in humans classified by their secondary structure content, fibrillar morphology by Bio-nanoimaging. http://dx.doi.org/10.1016/B978-0-12-394431-3.00007-9 Copyright © 2014 Elsevier Inc. All rights reserved.
Probes that Report their Conformation and Environment 71 Hydrophobic Probes and Molecular Rotors 71 Luminescent Conjugated Oligo- and Polythiophenes 73 Assessing Pathology Spread and Quantification 75 Applications and Implications of Polymorphism-Sensitive Reporters77 Diagnostics77 Therapeutics77
electron microscopy, and birefringent staining with the azo cotton dye, Congo Red. These features represent common ality among amyloid fibrils and define the current classifi cation criteria for an amyloid. More recently recognized is the ability of amyloid fibrils to propagate or seed by addition of cognate protein mono mers to fibril ends by a process of conformational conver sion, resulting in fibril growth. Initially observed as part of the infectivity process of prions, this templating activity, analogous to crystal seeding in which the structural/confor mational properties of the seed are faithfully replicated, is another common property of amyloid fibrils that has conse quences for pathology as well as for applications as nano materials and their toxicology.
LIGAND-BINDING PHENOTYPE Before Congo Red was discovered to characteristically stain amyloid fibrils with linear birefringence, Nature had evolved serum amyloid P protein (SAP), a calcium-dependent pentameric protein of the pentraxin family, which rec ognizes the common amyloid fibril structural fold and occurs bound to extracellular amyloid fibrils, perhaps to shield amyloid fibrils from toxic interactions or to possibly
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assist in their removal. Imaging with radiolabeled SAP injected into patients [2] reveals the distribution of amy loid fibril deposits in peripheral organs and connective tis sue and can be used to assess the effectiveness of therapies designed to remove the deposits.
Conformational/Configurational Antibodies Recently, a number of antibodies have been produced that recognize amyloid fibrils in a primary sequence-indepen dent fashion [3,4]. Other antibodies recognize only nonfibrillar oligomeric forms of amyloidogenic proteins and peptides but not the monomeric forms [5–11]. While these antibodies are believed to recognize conformational fea tures that differentiate among the multimeric species, the molecular bases of the epitopes remain to be defined for most antibodies. Radiolabeled SAP and antibodies have proven useful in peripheral applications outside of the brain, but they have limited ability to reach the brain. Imaging the amyloid lesions (Aβ, tau, α-synuclein) involved in neurodegenera tive diseases which account for the vast majority of cases of amyloidoses requires high-affinity small molecules that will penetrate the blood–brain barrier and, if not bound, can be rapidly cleared from the brain. High affinity is required to result in sufficient quantities of radioligand binding to the pathologic lesions at the nanomolar ligand concentra tions attainable in vivo for reliable quantification. The first clinically useful brain-imaging ligand was the uncharged thioflavin T analog [11] C-labeled benzothiazole Pitts burgh Compound B (PIB) introduced in 2002, followed by the more user-friendly longer radioactive half life [18] F-labeled stilbene derivatives Florbetapir (AV-45) (Amyvid, approved in 2012) and Florbetaben (filing was anticipated in the 3rd quarter of 2012).
Ligand-Binding Sites Analysis of the in vitro binding of different ligand structures to synthetic Aβ fibrils by Lockhart et al [12,13] indicated that there were distinct binding sites for ligands that could accommodate different chemical structures, and that the density of the different sites were different [BS3 (Me-BTA1) << BS1 (thioflavin T) < BS2 (Congo Red)]. The detailed molecular basis for these binding sites is unknown because of the difficulties obtaining high-resolution fibril structures, and modeling studies remain to be validated by experimen tal evidence. Congo Red sites (BS2) were the most abun dant, 1:1 with Aβ molecules in the fibrils. The other ligand sites on Aβ fibrils were less abundant. A key observation was that, although Congo Red (BS2) binding to synthetic Aβ fibrils remained fairly constant (CR/Aβ monomer), the fibrils induced the characteristic fluorescence change in thioflavin T (BS1) with an efficiency dependent on the fibril
formation conditions which also modulate fibril morphol ogy [14]. This suggested that the ThT binding and/or fluo rescent properties were sensitive to fibril polymorphism, with Congo Red less so. The potential disease relevance of Aβ fibril polymorphism was highlighted by the observation that PIB (BS3) binding density was very low in synthetic Aβ fibrils, and in animal models of Aβ pathology (trans genic mice [15] and (LeVine unpublished), nonhuman pri mates [16], and canines (LeVine, unpublished), but high in humans with AD and Down syndrome [17] despite similar amounts of Aβ amyloid pathology. A hypothesis that can be drawn from these observations is that specific polymorphic forms of Aβ amyloid (parenchymal and vascular) fibrils are related to disease status because only humans progress to dementia.
Peptides The selective templating of Aβ peptides by synthetic Aβ fibrils and AD Aβ pathology has permitted proof-of-concept labeling of Aβ pathology with tagged Aβ peptides in animal models [18,19], but the challenges of brain penetration with peptides and their derivatives compared to small-molecule ligands have hindered widespread application of this tech nology.
Cerebrovascular Amyloid Cerebrovascular accumulation of Aβ(1-40) (Aβ1-40) in the smooth-muscle layer around blood vessels (amyloid angi opathy) is associated with vessel fragility and small-vessel hemorrhage [20]. Vascular amyloid is comorbid with Aβ(1-42)-containing parenchymal Aβ plaques in AD, and it binds Congo Red and its analogs as well as the thiofla vines. A recent report identifies certain resorufin analogs as selective for cerebrovascular amyloid angiopathy over parenchymal Aβ deposits, occupying a distinct binding site. Current cerebrovascular-specific amyloid ligands lack sufficient affinity for in vivo imaging [21]. The molecular basis for this selectivity and its relationship to the BS(13) ligand binding sites on synthetic and parenchymal Aβ fibrils remains to be determined.
Imaging Non-Aβ Amyloid Brain Pathology Specific ligands that could distinguish individual amyloid proteins by in vivo imaging have been elusive. Consistent with general Congo Red binding to the defining cross-β sheet structure in amyloid fibrils, the BS2 site seems to be ubiquitously present on amyloid fibrils formed from a vari ety of proteins. The BS2 site is also present on neurofibril lary tau tangles which bind Congo Red and thioflavine S, commensurate with their amyloid character, but the affin ity and amount of tau lesions for PIB (BS3) are too low for imaging. Tau-selective ligands have been identified;
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however, they lack the requisite affinity and sufficient selectivity to distinguish tau from the more abundant Aβ in AD and in other mixed pathologies [22–25]. Similarly, α-synuclein cannot be imaged with PIB in humans [26]. Thus, high-affinity PIB binding is specific for Aβ fibrillar pathology of neurodegenerative diseases. Recently, Taghavi and co-workers [25] showed that the structure–activity relationship of N-benzylidene-benzohy drazide (NBB) binding to tau and Amyloid(1-42) fibrils indicated differential selectivity for these protein aggre gates. The NBB, BSc4014, demonstrated selective tangle staining, whereas NBB BSc3869 exhibits selective Aβ plaque staining.
PROBES THAT REPORT THEIR CONFORMATION AND ENVIRONMENT The most common small amyloid ligands are derivatives of Congo Red or the benzothiazole thioflavins. These probes are rather selective for protein aggregates having an exten sive content of cross β-pleated sheet conformation and structural regularity. However, it is evident that significant morphologic variation can exist between different amy loid fibrils formed from the same peptide or protein. Most conventional amyloid probes, with the exception of certain antibodies, do not distinguish protein aggregates of diverse morphologic origin at the light microscopic level. Recent evidence indicates that pre-fibrillar states preceding the for mation of well-defined amyloid fibrils are likely to play a critical role in the pathogenesis of protein aggregation dis eases [27–29]. The small-molecule-based amyloid ligands fail to recognize these pre-fibrillar species. Hence, there is a need for small-molecule amyloid ligands that identify a broader subset of proteinaceous aggregated species and variety of morphologically distinct protein aggregates.
Hydrophobic Probes and Molecular Rotors Many amyloid probe targeting studies have employed fluorescence spectroscopy to take advantage of the intrin sic sensitivity, applicability to histology, and potential for reporting the molecular environment of the probe. Several of the molecular scaffolds for in vitro or ex vivo detection have been developed for in vivo radioisotope PET or SPECT imaging of amyloid in the clinic (naphthalenes (FDDNP), styrylbenzenes (florbetapir, florbetaben), benzothiazoles (PIB)) (Fig. 7.2B). Amyloid probes are frequently based on substituted aro matic ring systems, such as benzophenoxazine, benzothiazole, benzoxazole, benzofuran, imidazopyridine, napthalene, stil bene, thiophene, or combinations thereof. Molecular probes with an extended linear configuration have been most widely studied. Their selectivity and affinity for amyloid are likely due to binding in deep elongated grooves in the amyloid fibril
FIGURE 7.1 High-resolution model of Congo Red bound to an amyloid fibril composed of the 218–289 fragment of HET-s. The fibril structure is rendered in light green and the Congo Red molecule as a space-filling model. The planarized Congo Red molecule binds in a hydrophobic groove aligned along the fibril axis. Specific alignment of the binding site was dictated by ionic bonds between the sulfonate side groups of Congo Red and the residue Lys229 in the amyloid fibril. The figure was made in PyMol using the pdb code 2LBU.
surface perpendicular to the fibril axis defined by the crossbeta sheet packing of the secondary structure as well as the zipper-like configuration of the constituent side-chains form ing the groove wall (Fig. 7.1). A high-resolution model of such a binding mode was recently presented for Congo Red bound to the yeast prion protein HET-s [30] which is suggested to be the BS2-type of binding site common to many different amy loid fibrils. The preference for linear probes binding to mature fibrils, rather than to early formed prefibrillar oligomers, is suggested by the fibril binding of ThT and 1-anilinonaphtha lene-8-sulfonate (ANS), while the fluorescence of the bulkier 4-(dicyanovinyl)-julolidine (DCVJ) and 4, 4-bis-1-phenylamino-8-naphthalene sulfonate (bis-ANS) increased in the presence of the prefibrillar oligomers [31] This observation suggests that selective prefibrillar probes should be less planar and less linear or possess a flexible backbone (see LCO sec tion below) to accommodate such configurations. The physicochemical basis for the increased fluores cence of molecular probes upon binding to prefibrillar states and to amyloid fibrils can be attributed mainly to two effects: (1) intrinsic molecular quenching, and (2) sol vent quenching and polarization. Molecular rotors (DDNP, DCVJ and NIAD-4, ANCA) (Fig. 7.2) are intrinsically
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FIGURE 7.2 (A) Chemical structures of small hydrophobic probes and molecular rotors. (B) Chemical structures of amyloid ligands.
Chapter | 7 Polymorphism Reporters
quenched in solution due to rapid rotation of quenching sub stituents. Steric restriction upon binding to an oligomeric state or to an amyloid fibril reduces the quenching. The amyloid fibril-induced fluorescence of ThT is suggested to occur by this mechanism [31]. Solvent quenching or polar ization of excited-state probes which are readily polarizable (CRANAD-2, ANS, NIAD-4, bis-ANS, methoxy-X04, X-34, and Nile Red) (Fig. 7.2) are stabilized by water but are much less affected by a hydrophobic milieu. A small Stokes shift with an enhanced fluorescence quantum yield is observed in hydrophobic environments. An example of a probe reporting molecular character istics of the ligand-binding sites on amyloid fibrils is the benzophenoxazine-based dye Nile Red, which can eluci date the local polarity of its fibril binding site in solution for several amyloidogenic proteins (Aβ, insulin, lysozyme, prion protein, and transthyretin [32]). The dielectric con stant for the binding pocket was estimated by calibrating the fluorescence of unbound probe in a series of organic solvents. Some of the amyloid fibril binding sites for Nile Red were more hydrophobic than octanol, a solvent mod eling the dielectric of the hydrophobic core of globular proteins. The structural similarity between Nile Red and extended amyloid probes, as well as partial competition with ThT [32], suggests that its binding site is BS2 and/or BS1. Nile Red does not change fluorescence in the pres ence of oligomeric prefibrillar states, instead targeting more mature amyloid fibrils. Unfortunately, Nile Red is too lipophilic for specific binding to amyloid in complex tissue environments, a property exploited in its use as a histologi cal lipid probe [33]. Aminonaphthlanene 2-cyanoacrylate (ANCA) is an example of a class of probes that, like Nile Red, can dis tinguish differences in amyloid binding-site polarity, but it appears to be selective enough for amyloid imaging in tissue sections [34]. The ANCA class of probes displays an amy loid fibril-induced molecular rotor fluorescence response (intrinsic quenching and polarizability). Aggregated states of proteins which have not attained the final mature form of an amyloid fibril are large com pared to soluble globular proteins or unfolded peptides and they display exposed hydrophobic patches, characteristics addressable for selective probe targeting. Bulky and flexible molecules are more likely than linear structures to detect these species. ANS has been reported to detect cytotoxic oligomeric states in vitro [35]. The authors suggest that the amount of exposed organized hydrophobic surface of the oligomers quantified by ANS binding is correlated with cytotoxicity due to disturbance of the membrane bilayer or membrane proteins. Probes that can directly delineate surface characteristics of amyloidogenic states in vivo will advance future research, and will be applicable to diagnos tics and therapeutics.
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Luminescent Conjugated Oligo- and Polythiophenes Luminescent conjugated polythiophenes (LCPs) with molecular scaffolds consisting of polymers of thiophene moieties have evolved as an interesting and uniquely use ful class of fluorescent probes. These molecules provide a distinctive optical read-out through the impact of detailed biomolecular interactions on the conformation and the geometry of the LCP backbone [36–39]. The structurally induced optical changes of the LCP backbone also allow the tantalizing prospect of detection of altered conforma tions of biomolecules. A variety of LCP-based amyloidspecific ligands have been reported (Fig. 7.3A) [40–42]. These ligands provide detailed morphology of the protein deposits and conformational phenotyping of distinct and polymorphic protein aggregates. Beyond reporting the total amount, the LCPs allow spectral fingerprinting of polymor phic populations of these assemblies. These ligands have proved effective for studying protein aggregates ex vivo and in vivo in transgenic mouse models with AD Aβ pathol ogy [42,43], in transgenic mice infected with distinct prion strains [44–48], and in human clinical tissue samples [49]. Application of LCPs to transgenic mouse models with AD Aβ pathology revealed a striking heterogeneity in the characteristic plaques composed of the Aβ peptide [42]. LCP staining of brain tissue slices revealed distinct subpopulations of plaques, seen as plaques fluorescing different colors, and intraplaque heterogeneity of color. The spectral features of LCPs are useful for comparison of polymorphic protein aggregates in well-defined experimental systems. Polymorphic protein aggregates are found in many protein aggregation disorders, and this is especially notable in the infectious prion diseases. Prions can occur as differ ent strains that are most likely encoded in the tertiary or quaternary structure of the prion aggregates. LCP staining of protein aggregates in brain sections from mice infected with distinct prion strains exhibited a distinct spectroscopic signature when bound to strain-specific prion deposits [44]. Different prion strains could also be distinguished by their staining selectivity by LCPs with distinct ionic side chains. The spectral differences due to different thiophene back bone conformations for the anionic LCP, polythiophene acetic acid (PTAA), were visualized by correlation plots of the ratios of the intensities of fluorescence emission at specific wavelengths. Prion aggregates associated with distinct prion strains, chronic wasting disease (CWD), and sheep scrapie, were easily distinguished (Fig. 7.3B). LCP fluorescence analysis has also been used to identify novel prion strains [45] and to investigate the molecular basis for interspecies prion disease transmission in mice [46,47]. PTAA allows ready characterization and identification of mixed prion strains within a single host (Fig. 7.3C) [48].
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FIGURE 7.3 (A) Chemical structures of luminescent conjugated polythiophenes that have been utilized as amyloid ligands. (B) Fluorescence images and spectra (right) of prion deposits, chronic wasting disease (mCWD) (left) and sheep scrapie (mNS) (middle) stained by PTAA. Some representative PTAA-stained prion deposits are indicated by white arrows. (C) Fluorescence images of PTAA stained mCWD and mNS deposits in a single host. mCWD deposits have a green spectrum (middle), whereas mNS deposits have a red spectrum (right). (D) Fluorescence image of PTAA bound to amyloid deposits in tissue samples, heart (left) and liver (right) from a patient diagnosed with AA-amyloidosis. Two different colors are observed from distinct deposits, suggesting the presence of polymorphic AA-amyloid.
The presence of multiple prion strains is difficult to demon strate by conventional biochemical methods. Studies of prion deposits in tissue samples [44–48] sug gest that the LCP emission profile is an indirect readout of structural difference between prion deposits associated with
distinct prion strains. To mechanistically relate geometri cal alterations of the LCPs to structural variations of the protein deposits, rather than their composition, studies were carried out with recombinant prion protein, PrP, [44,50,51]. Different forms of in vitro amyloid fibrils generated from
Chapter | 7 Polymorphism Reporters
recombinant mouse, Syrian hamster or human prion pro tein (mPrP, sHaPrP, HuPrP) displayed distinct LCP spec tra. The emission profile of PTAA distinguished individual aggregates within different PrP fibril preparations that were chemically identical (same PrP sequence). Thus, spectral differences in tissue observed for PTAA were most likely due to structural differences between the fibrils. LCP fin gerprinting will be useful for the analysis of conformational polymorphism in other protein aggregation diseases, as similar phenomena may be much more frequent in other neurodegenerative protein aggregation disorders and amy loidoses [52]. PTAA emission spectral profiles can be used to sub-type systemic amyloidoses in tissue samples similar to prion strains [44]; PTAA staining revealed the existence of multiple types of AA amyloid in a single host (Fig. 7.3D) [49]. Although LCPs are uniquely suited to spectroscopic characterization of polymorphic amyloid deposits, they do not detect pre-fibrillar aggregates preceding the forma tion of mature amyloid fibrils. They are also relatively high molecular weight, polydisperse, and do not penetrate the blood–brain barrier for in vivo imaging of protein aggre gates in the brain. In 2009, Åslund and co-workers [53] introduced a novel class of smaller chemically defined LCPs, denoted luminescent conjugated oligothiophenes (LCOs), based on a pentameric thiophene backbone (Fig. 7.4A). These molecules are amyloid-specific ligands under physiologic conditions and show striking enhanced fluo rescence and distinct emission wavelength profiles when bound to protein aggregates associated with AD and prion diseases. The LCOs display a distinct emission profile with well-resolved sub-structure upon binding to recombinant Aβ(1-42) amyloid fibrils, implying that the backbone of the LCOs becomes more rigid upon binding to the fibrils (Fig. 7.4B). Both thioflavin T (ThT) and one of the LCOs, p-FTAA, reported conventional nucleated fibrillation kinetic behavior of recombinant Aβ(1-42) peptide, including a lag phase, a rapid exponential growth phase, and a final plateau phase. However, with the recombinant Aβ(1-40) peptide, pFTAA fluorescence revealed an earlier growth phase than ThT, indicating that p-FTAA detected pre-fibrillar Aβ(140) species preceding the formation of amyloid fibrils (Fig. 7.4B). p-FTAA also detects non-thioflavinophilic pre-fibril lar aggregates in vitro for a variety of other amyloidogenic proteins, including PrP, insulin, lysozyme and different Aβ peptides [54]. Using a small library of thiophenes with distinct chain length, Nilsson and co-workers were able to show that a thiophene backbone consisting of at least five thiophene units was necessary to detect the pre-fibrillar aggregated species [55]. The high selectivity for protein aggregates and the distinctive conformation-induced optical properties of the novel chemically defined LCOs were further demon strated when applied to cryo-sectioned brain tissue from
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AD patients (Fig. 7.4C). The major pathologic hallmarks of AD, Aβ deposits, neurofibrillary tangles (NFTs) and dystrophic neurites, were clearly detected by all of the LCOs. Moreover, the LCOs showed complete co-localiza tion with 6E10 and AT8, antibodies conventionally used to stain Aβ and phosphorylated tau in NFTs, respectively [53,55]. Pentameric, hexameric, and heptameric LCOs with the terminal thiophenes substituted with carboxyl groups at the α-position (Fig. 7.4A) gave different emis sion spectra when bound to the Aβ and tau pathologies in the human AD brain. This novel class of oligothiophenebased amyloid-specific dyes is a promising histologic tool for spectral assignment of distinct protein aggregates observed in AD. Surprisingly, anionic LCOs with four negative charges can be used for in vivo optical imaging of protein aggregates (Fig. 7.4D) [53]. The labeling of plaques in the transgenic APP/PS1 mouse brain can be observed in real time by mul tiphoton microscopy through a cranial window overlying the parietal cortex. The staining was persistent as individ ual LCO stained amyloid deposits were detectable even 1 week post-injection of the dye. p-FTAA also specifically labels prion deposits associated with distinct prion strains in vivo, and the strains could be distinguished by their p-FTAA spectral signature [55]. Lately, it was also shown that the heptameric LCO, h-FTAA, could be utilized for spectral assignment of CβAA, Aβ plaques and intracellular tau aggregates in transgenic mice after a single intravenous injection of the LCO [56]. Although the conformation-dependent spectral informa tion would be lost, radiolabeled versions of the oligomeric thiophenes for clinical PET imaging could be developed. Optimization of the thiophene core structure will be required to provide the requisite binding selectivities for the different protein pathologies, the different species of protein aggre gates (oligomers, fibrils), and their polymorphic forms.
ASSESSING PATHOLOGY SPREAD AND QUANTIFICATION The dissemination of misfolded protein pathology and neu ronal dysfunction of the major neurodegenerative diseases follows stereotypical patterns through the brain. While the specific brain regions involved differ between diseases, in keeping with their clinical presentation the spreading is reminiscent of the neuronal connectivity between the affected regions. The proposed mechanism is the transport of fragments or nuclei of misfolded Aβ, tau, or α-synuclein pathology within neurons which template fibril or soluble assembly growth from cognate monomeric species. Exocy tosis of misfolded seeds in exosomes and their uptake by other neurons has also been observed [57,58] and may con stitute another mechanism that could also account for the spread of pathology to non-connected brain regions.
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FIGURE 7.4 (A) Chemical structures of tetrameric (q-FTAA), pentameric (p-FTAA) and heptameric (h-FTAA) luminescent conjugated oligothiophenes that have been utilized as amyloid ligands. (B) Fluorescence spectrum (left) of p-FTAA mixed with recombinant Aβ(1-40) amyloid fibrils (green) or freshly dissolved Aβ(1-40) (blue). Comparison between ThT and p-FTAA for monitoring the kinetics of recombinant Aβ(1-40) amyloid fibril formation. Notably, p-FTAA reacts earlier than ThT. (C) Fluorescence image (left) and spectra (right) of p-FTAA bound to the two pathologic hallmarks, Aβ deposits (green) and neurofibrillary tangles (yellow-red), in brain tissue samples from an AD patient. (D) Fluorescence images after a single intravenous injection of hFTAA in transgenic mice. Characteristic amyloid lesions, cerebrovascular β-amyloid angiopathy (CβAA), Aβ plaques, and intracellular tau aggregates are labeled throughout the brain and visualized by green fluorescence from h-FTAA.
The fidelity of template structural features is frequently very high, restricting seeding mainly to the cognate peptide, although this can be altered in vitro, and there is some evi dence for direct or indirect heterologous templating between
pathologies [59]. The templating fidelity extends to polymor phic structural variants which are observable in the form of prion strains with different characteristics which can co-exist in the brains of infected animals but replicate independently
Chapter | 7 Polymorphism Reporters
[48,60]. This property is exploited to amplify specific confor mations of prions in analytical methods such as protein mis folding cyclic amplification (PMCA) [61] or to template the formation of sufficient quantities of a particular fibril poly morph for structural analysis from a biologic sample from a synthetic or recombinant monomer [62]. The persistence of in vivo pFTAA staining of amyloid deposits could be utilized to trace dispersal in animal models in which spectrally dis tinct LCOs could be applied as amyloid ‘time stamps’.
APPLICATIONS AND IMPLICATIONS OF POLYMORPHISM-SENSITIVE REPORTERS Diagnostics It is clear from the increasing scope and evolving polymor phic complexity of misfolded protein proteopathies that the original tools for diagnosis, Congo Red staining and bire fringence and silver stains for Aβ plaques and tau, cannot provide the depth and wealth of information that may be required for accurate early detection of the disease process and for probing the underlying biology. There remains a dearth of ligands specific for soluble oligomeric species of misfolded proteins. All pathologic lesions of the same type are not equal in their impact on, or reflection of, the disease. Newer probes, such as the Aβ fibril binding site subtype-specific ligands and the poly- and oligothiophene fluorophores, are beginning to fill this gap. Multiplexed mixtures of probes, or at least multiple determinations, may be required. The true impact of these new probes remains to be established as they are in an early stage of development and require further validation at the pathologic level.
Therapeutics Certainly by their diagnostic impact, the new probes can assist in determining whether particular therapeutic strate gies have had the anticipated effect on specific polymorphic forms of protein aggregates. Whether by their direct interac tion with misfolded protein pathologies similar molecules could induce the dissociation or detoxification of polymor phic bioactive species awaits to be conclusively determined. There have been reports that affording amyloid targeting, e.g. with the polyphenolic compound curcumin, which is structurally similar to the amyloid reporters described in this chapter, increases the rate of conversion from the putatively neurotoxic prefibrillar oligomer assemblies into mature amyloid fibrils, thereby reducing neurotoxicity in transgenic Drosophila expressing Aβ peptides [63]. A similar mechanism was reported for the curcumin-related Orcein class of small molecules, suggesting a commonal ity [64]. Testing a library of LCP molecules as therapeu tic agents against prions in organotypic cultured cerebellar slices revealed a dramatic structure-dependent differential
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reduction of infectivity while accumulated protease- resistant PrP increased [65]. This showed that LCP-induced hyperstabilization of PrP amyloid fibrils was an operational trapping mechanism for reducing infectivity by suppressing fragmentation. Selectively targeting specific polymorphic forms of amyloid may have potential therapeutic benefit for neurotoxic amyloidogenic proteins such as Aβ and PrP.
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