Novel <i>trans</i>-Stilbene-based Fluorophores as Probes for Spectral Discrimination of Native and Protofibrillar Transthyretin

Accumulation of misfolded transthyretin (TTR) as amyloid fibrils causes various human disorders. Native transthyretin is a neurotrophic protein and is a putative extracellular molecular chaperone. Several fluorophores have been shown <i>in vitro</i> to bind selectively to native TTR. Other compounds, such as thioflavin T, bind TTR amyloid fibrils. The probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to both native and fibrillar TTR, becoming highly fluorescent, but with indistinguishable emission spectra for native and fibrillar TTR. Herein we report our efforts to develop a fluorescent small molecule capable of binding both native and misfolded protofibrillar TTR, providing distinguishable emission spectra. We used microwave synthesis for efficient production of a small library of <i>trans</i>-stilbenes and fluorescence spectral screening of their binding properties. We synthesized and tested 22 <i>trans</i>-stilbenes displaying a variety of functional groups. We successfully developed two naphthyl-based <i>trans</i>-stilbenes probes that detect both TTR states at physiological concentrations. The compounds bound with nanomolar to micromolar affinities and displayed distinct emission maxima upon binding native or misfolded protofibrillar TTR (>100 nm difference). The probes were mainly responsive to environment polarity providing evidence for the divergent hydrophobic structure of the binding sites of these protein conformational states. Furthermore, we were able to successfully use one of these probes to quantify the relative amounts of native and protofibrillar TTR in a dynamic equilibrium. In conclusion, we identified two <i>trans</i>-stilbene-based fluorescent probes, (<i>E</i>)-4-(2-(naphthalen-1-yl)­vinyl)­benzene-1,2-diol (<b>11</b>) and (<i>E</i>)-4-(2-(naphthalen-2-yl)­vinyl)­benzene-1,2-diol (<b>14</b>), that bind native and protofibrillar TTR, providing a wide difference in emission maxima allowing conformational discrimination by fluorescence spectroscopy. We expect these novel molecules to serve as important chemical biology research tools in studies of TTR folding and misfolding

Derivatization of a Bioorthogonal Protected Trisaccharide LinkerToward Multimodal Tools for Chemical Biology

When cross-linking biomolecules to surfaces or to other biomolecules, the use of appropriate spacer molecules is of great importance. Mimicking the naturally occurring spacer molecules will give further insight into their role and function, possibly unveil important issues regarding the importance of the specificity of carbohydrate-based anchor moieties, in e.g., glycoproteins and glycosylphosphatidylinositols. Herein, we present the synthesis of a lactoside-based trisaccharide, potentially suitable as a heterobifunctional bioorthogonal linker molecule whereon valuable chemical handles have been conjugated. An amino-derivative having thiol functionality shows promise as novel SPR-surfaces. Furthermore, the trisaccharide has been conjugated to a cholesterol moiety in combination with a fluorophore which successfully assemble on the cell surface in lipid microdomains, possibly lipid-rafts. Finally, a Cu^I-catalyzed azide–alkyne cycloaddition reaction (CuAAC) confirms the potential use of oligosaccharides as bioorthogonal linkers in chemical biology

Divergent Age-Dependent Conformational Rearrangement within Aβ Amyloid Deposits in APP23, APPPS1, and <i>App</i>^{<i>NL‑F</i>} Mice

Amyloid plaques composed of fibrils of misfolded Aβ peptides are pathological hallmarks of Alzheimer’s disease (AD). Aβ fibrils are polymorphic in their tertiary and quaternary molecular structures. This structural polymorphism may carry different pathologic potencies and can putatively contribute to clinical phenotypes of AD. Therefore, mapping of structural polymorphism of Aβ fibrils and structural evolution over time is valuable to understanding disease mechanisms. Here, we investigated how Aβ fibril structures in situ differ in Aβ plaque of different mouse models expressing familial mutations in the AβPP gene. We imaged frozen brains with a combination of conformation-sensitive luminescent conjugated oligothiophene (LCO) ligands and Aβ-specific antibodies. LCO fluorescence mapping revealed that mouse models APP23, APPPS1, and AppNL‑F have different fibril structures within Aβ-amyloid plaques depending on the AβPP-processing genotype. Co-staining with Aβ-specific antibodies showed that individual plaques from APP23 mice expressing AβPP Swedish mutation have two distinct fibril polymorph regions of core and corona. The plaque core is predominantly composed of compact Aβ40 fibrils, and the corona region is dominated by diffusely packed Aβ40 fibrils. Conversely, the AβPP knock-in mouse AppNL‑F, expressing the AβPP Iberian mutation along with Swedish mutation has tiny, cored plaques consisting mainly of compact Aβ42 fibrils, vastly different from APP23 even at elevated age up to 21 months. Age-dependent polymorph rearrangement of plaque cores observed for APP23 and APPPS1 mice >12 months, appears strongly promoted by Aβ40 and was hence minuscule in AppNL‑F. These structural studies of amyloid plaques in situ can map disease-relevant fibril polymorph distributions to guide the design of diagnostic and therapeutic molecules

Multimodal Chemical Imaging of Amyloid Plaque Polymorphism Reveals Aβ Aggregation Dependent Anionic Lipid Accumulations and Metabolism

Amyloid plaque formation constitutes one of the main pathological hallmarks of Alzheimer’s disease (AD) and is suggested to be a critical factor driving disease pathogenesis. Interestingly, in patients that display amyloid pathology but remain cognitively normal, Aβ deposits are predominantly of diffuse morphology suggesting that cored plaque formation is primarily associated with cognitive deterioration and AD pathogenesis. Little is known about the molecular mechanism responsible for conversion of monomeric Aβ into neurotoxic aggregates and the predominantly cored deposits observed in AD. The structural diversity among Aβ plaques, including cored/compact- and diffuse, may be linked to their distinct Aβ profile and other chemical species including neuronal lipids. We developed a novel, chemical imaging paradigm combining matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) and fluorescent amyloid staining. This multimodal imaging approach was used to probe the lipid chemistry associated with structural plaque heterogeneity in transgenic AD mice (tgAPP_Swe) and was correlated to Aβ profiles determined by subsequent laser microdissection and immunoprecipitation-mass spectrometry. Multivariate image analysis revealed an inverse localization of ceramides and their matching metabolites to diffuse and cored structures within single plaques, respectively. Moreover, phosphatidylinositols implicated in AD pathogenesis, were found to localize to the diffuse Aβ structures and correlate with Aβ1–42. Further, lysophospholipids implicated in neuroinflammation were increased in all Aβ deposits. The results support previous clinical findings on the importance of lipid disturbances in AD pathophysiology and associated sphingolipid processing. These data highlight the potential of multimodal imaging as a powerful technology to probe neuropathological mechanisms

¹¹C and ¹⁸F Radiolabeling of Tetra- and Pentathiophenes as PET-Ligands for Amyloid Protein Aggregates

Three oligothiophenes were evaluated as PET ligands for the study of local and systemic amyloidosis <i>ex vivo</i> using tissue from patients with amyloid deposits and <i>in vivo</i> using healthy animals and PET-CT. The <i>ex vivo</i> binding studies revealed that all three labeled compounds bound specifically to human amyloid deposits. Specific binding was found in the heart, kidney, liver, and spleen. To verify the specificity of the oligothiophenes toward amyloid deposits, tissue sections with amyloid pathology were stained using the fluorescence exhibited by the compounds and evaluated with multiphoton microscopy. Furthermore, a <i>in vivo</i> monkey PET-CT study showed very low uptake in the brain, pancreas, and heart of the healthy animal indicating low nonspecific binding to healthy tissue. The biological evaluations indicated that this is a promising group of compounds for the visualization of systemic and localized amyloidosis

Binding of Polythiophenes to Amyloids: Structural Mapping of the Pharmacophore

Luminescent conjugated polythiophenes bind to amyloid proteins with high affinity. Their fluorescence properties, which are modulated by the detailed conformation in the bound state, are highly sensitive to structural features of the amyloid. Polythiophenes therefore represent diagnostic markers for the detection and differentiation of pathological amyloid aggregates. We clarify the binding site and mode of two different polythiophenes to fibrils of the prion domain of the HET-s protein by solid-state NMR and correlate these findings with their fluorescence properties. We demonstrate how amyloid dyes recognize distinct binding sites with specific topological features. Regularly spaced surface charge patterns and well-accessible grooves on the fibril surface define the pharmacophore of the amyloid, which in turn determines the binding mode and fluorescence wavelength of the polythiophene

Evidence for Age-Dependent <i>in Vivo</i> Conformational Rearrangement within Aβ Amyloid Deposits

Deposition of aggregated Aβ peptide in the brain is one of the major hallmarks of Alzheimer’s disease. Using a combination of two structurally different, but related, hypersensitive fluorescent amyloid markers, LCOs, reporting on separate ultrastructural elements, we show that conformational rearrangement occurs within Aβ plaques of transgenic mouse models as the animals age. This important mechanistic insight should aid the design and evaluation of experiments currently using plaque load as readout

Evidence for Age-Dependent <i>in Vivo</i> Conformational Rearrangement within Aβ Amyloid Deposits

Deposition of aggregated Aβ peptide in the brain is one of the major hallmarks of Alzheimer’s disease. Using a combination of two structurally different, but related, hypersensitive fluorescent amyloid markers, LCOs, reporting on separate ultrastructural elements, we show that conformational rearrangement occurs within Aβ plaques of transgenic mouse models as the animals age. This important mechanistic insight should aid the design and evaluation of experiments currently using plaque load as readout