Thiophene-Based Ligands for Specific Assignment of Distinct Aß Pathologies in Alzheimer's Disease.

Aggregated species of amyloid-ß (Aß) are one of the pathological hallmarks in Alzheimer's disease (AD), and ligands that selectively target different Aß deposits are of great interest. In this study, fluorescent thiophene-based ligands have been used to illustrate the features of different types of Aß deposits found in AD brain tissue. A dual-staining protocol based on two ligands, HS-276 and LL-1, with different photophysical and binding properties, was developed and applied on brain tissue sections from patients affected by sporadic AD or familial AD associated with the PSEN1 A431E mutation. When binding to Aß deposits, the ligands could easily be distinguished for their different fluorescence, and distinct staining patterns were revealed for these two types of AD. In sporadic AD, HS-276 consistently labeled all immunopositive Aß plaques, whereas LL-1 mainly stained cored and neuritic Aß deposits. In the PSEN1 A431E cases, each ligand was binding to specific types of Aß plaques. The ligand-labeled Aß deposits were localized in distinct cortical layers, and a laminar staining pattern could be seen. Biochemical characterization of the Aß aggregates in the individual layers also showed that the variation of ligand binding properties was associated with certain Aß peptide signatures. For the PSEN1 A431E cases, it was concluded that LL-1 was binding to cotton wool plaques, whereas HS-276 mainly stained diffuse Aß deposits. Overall, our findings showed that a combination of ligands was essential to identify distinct aggregated Aß species associated with different forms of AD.

Real-time imaging of mitochondrial redox reveals increased mitochondrial oxidative stress associated with amyloid ß aggregates in vivo in a mouse model of Alzheimer's disease.

BACKGROUND: Reactive oxidative stress is a critical player in the amyloid beta (Aß) toxicity that contributes to neurodegeneration in Alzheimer's disease (AD). Damaged mitochondria are one of the main sources of reactive oxygen species and accumulate in Aß plaque-associated dystrophic neurites in the AD brain. Although Aß causes neuronal mitochondria reactive oxidative stress in vitro, this has never been directly observed in vivo in the living mouse brain. Here, we tested for the first time whether Aß plaques and soluble Aß oligomers induce mitochondrial oxidative stress in surrounding neurons in vivo, and whether this neurotoxic effect can be abrogated using mitochondrial-targeted antioxidants. METHODS: We expressed a genetically encoded fluorescent ratiometric mitochondria-targeted reporter of oxidative stress in mouse models of the disease and performed intravital multiphoton microscopy of neuronal mitochondria and Aß plaques. RESULTS: For the first time, we demonstrated by direct observation in the living mouse brain exacerbated mitochondrial oxidative stress in neurons after both Aß plaque deposition and direct application of soluble oligomeric Aß onto the brain, and determined the most likely pathological sequence of events leading to oxidative stress in vivo. Oxidative stress could be inhibited by both blocking calcium influx into mitochondria and treating with the mitochondria-targeted antioxidant SS31. Remarkably, the latter ameliorated plaque-associated dystrophic neurites without impacting Aß plaque burden. CONCLUSIONS: Considering these results, combination of mitochondria-targeted compounds with other anti-amyloid beta or anti-tau therapies hold promise as neuroprotective drugs for the prevention and/or treatment of AD.

Two structurally defined Aß polymorphs promote different pathological changes in susceptible mice.

Misfolded Aß is involved in the progression of Alzheimer's disease (AD). However, the role of its polymorphic variants or conformational strains in AD pathogenesis is not fully understood. Here, we study the seeding properties of two structurally defined synthetic misfolded Aß strains (termed 2F and 3F) using in vitro and in vivo assays. We show that 2F and 3F strains differ in their biochemical properties, including resistance to proteolysis, binding to strain-specific dyes, and in vitro seeding. Injection of these strains into a transgenic mouse model produces different pathological features, namely different rates of aggregation, formation of different plaque types, tropism to specific brain regions, differential recruitment of Aß40 /Aß42 peptides, and induction of microglial and astroglial responses. Importantly, the aggregates induced by 2F and 3F are structurally different as determined by ssNMR. Our study analyzes the biological properties of purified Aß polymorphs that have been characterized at the atomic resolution level and provides relevant information on the pathological significance of misfolded Aß strains.

Thiophene-Based Ligands for Histological Multiplex Spectral Detection of Distinct Protein Aggregates in Alzheimer's Disease.

The aggregation and accumulation of proteins in the brain is the defining feature of many devastating neurodegenerative diseases. The development of fluorescent ligands that bind to these accumulations, or deposits, is essential for the characterization of these neuropathological lesions. We report the synthesis of donor-acceptor-donor (D-A-D) thiophene-based ligands with different emission properties. The D-A-D ligands displayed selectivity towards distinct disease-associated protein deposits in histological sections from postmortem brain tissue of individuals affected by Alzheimer's disease (AD). The ability of the ligands to selectively identify AD-associated pathological alterations, such as deposits composed of aggregates of the amyloid-ß (Aß) peptide or tau, was reduced when the chemical composition of the ligands was altered. When combining the D-A-D ligands with conventional thiophene-based ligands, superior spectral separation of distinct protein aggregates in AD tissue sections was obtained. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between aggregated proteinaceous species, as well as offer novel strategies for developing multiplex fluorescence detection of protein aggregates in tissue sections.

Whole-brain microscopy reveals distinct temporal and spatial efficacy of anti-Aß therapies.

Many efforts targeting amyloid-ß (Aß) plaques for the treatment of Alzheimer's Disease thus far have resulted in failures during clinical trials. Regional and temporal heterogeneity of efficacy and dependence on plaque maturity may have contributed to these disappointing outcomes. In this study, we mapped the regional and temporal specificity of various anti-Aß treatments through high-resolution light-sheet imaging of electrophoretically cleared brains. We assessed the effect on amyloid plaque formation and growth in Thy1-APP/PS1 mice subjected to ß-secretase inhibitors, polythiophenes, or anti-Aß antibodies. Each treatment showed unique spatiotemporal Aß clearance, with polythiophenes emerging as a potent anti-Aß compound. Furthermore, aligning with a spatial-transcriptomic atlas revealed transcripts that correlate with the efficacy of each Aß therapy. As observed in this study, there is a striking dependence of specific treatments on the location and maturity of Aß plaques. This may also contribute to the clinical trial failures of Aß-therapies, suggesting that combinatorial regimens may be significantly more effective in clearing amyloid deposition.

Distinct Heterocyclic Moieties Govern the Selectivity of Thiophene-Vinylene-Based Ligands Towards Aß or Tau Pathology in Alzheime's Disease.

Distinct aggregated proteins are correlated with numerous neurodegenerative diseases and the development of ligands that selectively detect these pathological hallmarks is vital. Recently, the synthesis of thiophene-based optical ligands, denoted bi-thiophene-vinyl-benzothiazoles (bTVBTs), that could be utilized for selective assignment of tau pathology in brain tissue with Alzheime's disease (AD) pathology, was reported. Herein, we investigate the ability of these ligands to selectively distinguish tau deposits from aggregated amyloid-ß (Aß), the second AD associated pathological hallmark, when replacing the terminal thiophene moiety with other heterocyclic motifs. The selectivity for tau pathology was reduced when introducing specific heterocyclic motifs, verifying that specific molecular interactions between the ligands and the aggregates are necessary for selective detection of tau deposits. In addition, ligands having certain heterocyclic moieties attached to the central thiophene-vinylene building block displayed selectivity to aggregated Aß pathology. Our findings provide chemical insights for the development of ligands that can distinguish between aggregated proteinaceous species consisting of different proteins and might also aid in creating novel agents for clinical imaging of tau pathology in AD.

Structural Properties Dictating Selective Optotracer Detection of Staphylococcus aureus.

Optotracers are conformation-sensitive fluorescent tracer molecules that detect peptide- and carbohydrate-based biopolymers. Their binding to bacterial cell walls allows selective detection and visualisation of Staphylococcus aureus (S. aureus). Here, we investigated the structural properties providing optimal detection of S. aureus. We quantified spectral shifts and fluorescence intensity in mixes of bacteria and optotracers, using automatic peak analysis, cross-correlation, and area-under-curve analysis. We found that the length of the conjugated backbone and the number of charged groups, but not their distribution, are important factors for selective detection of S. aureus. The photophysical properties of optotracers were greatly improved by incorporating a donor-acceptor-donor (D-A-D)-type motif in the conjugated backbone. With significantly reduced background and binding-induced on-switch of fluorescence, these optotracers enabled real-time recordings of S. aureus growth. Collectively, this demonstrates that chemical structure and photophysics are key tunable characteristics in the development of optotracers for selective detection of bacterial species.

Synapsin III gene silencing redeems alpha-synuclein transgenic mice from Parkinson's disease-like phenotype.

Fibrillary aggregated α-synuclein (α-syn) deposition in Lewy bodies (LB) characterizes Parkinson's disease (PD) and is believed to trigger dopaminergic synaptic failure and a retrograde terminal-to-cell body neuronal degeneration. We described that the neuronal phosphoprotein synapsin III (Syn III) cooperates with α-syn to regulate dopamine (DA) release and can be found in the insoluble α-syn fibrils composing LB. Moreover, we showed that α-syn aggregates deposition, and the associated onset of synaptic deficits and neuronal degeneration occurring following adeno-associated viral vectors-mediated overexpression of human α-syn in the nigrostriatal system are hindered in Syn III knock out mice. This supports that Syn III facilitates α-syn aggregation. Here, in an interventional experimental design, we found that by inducing the gene silencing of Syn III in human α-syn transgenic mice at PD-like stage with advanced α-syn aggregation and overt striatal synaptic failure, we could lower α-syn aggregates and striatal fibers loss. In parallel, we observed recovery from synaptic vesicles clumping, DA release failure, and motor functions impairment. This supports that Syn III consolidates α-syn aggregates, while its downregulation enables their reduction and redeems the PD-like phenotype. Strategies targeting Syn III could thus constitute a therapeutic option for PD.

Thiophene-Based Optical Ligands That Selectively Detect Aß Pathology in Alzheimer's Disease.

In several neurodegenerative diseases, the presence of aggregates of specific proteins in the brain is a significant pathological hallmark; thus, developing ligands able to bind to the aggregated proteins is essential for any effort related to imaging and therapeutics. Here we report the synthesis of thiophene-based ligands containing nitrogen heterocycles. The ligands selectively recognized amyloid-ß (Aß) aggregates in brain tissue from individuals diagnosed neuropathologically as having Alzheimer's disease (AD). The selectivity for Aß was dependent on the position of nitrogen in the heterocyclic compounds, and the ability to bind Aß was shown to be reduced when introducing anionic substituents on the thiophene backbone. Our findings provide the structural and functional basis for the development of ligands that can differentiate between aggregated proteinaceous species comprised of distinct proteins. These ligands might also be powerful tools for studying the pathogenesis of Aß aggregation and for designing molecules for imaging of Aß pathology.

Deciphering the Electronic Transitions of Thiophene-Based Donor-Acceptor-Donor Pentameric Ligands Utilized for Multimodal Fluorescence Microscopy of Protein Aggregates.

Anionic pentameric thiophene acetates can be used for fluorescence detection and diagnosis of protein amyloid aggregates. Replacing the central thiophene unit by benzothiadiazole (BTD) or quinoxaline (QX) leads to large emission shifts and basic spectral features have been reported [Chem. Eur. J. 2015, 21, 15133-13137]. Here we present new detailed experimental results of solvent effects, time-resolved fluorescence and examples employing multi-photon microscopy and lifetime imaging. Quantum chemical response calculations elucidate how the introduction of the BTD/QX groups changes the electronic states and emissions. The dramatic red-shift follows an increased conjugation and quinoid character of the π-electrons of the thiophene backbone. An efficient charge transfer in the excited states S1 and S2 compared to the all-thiophene analogue makes these more sensitive to the polarity and quenching by the solvent. Taken together, the results guide in the interpretation of images of stained Alzheimer disease brain sections employing advanced fluorescence microscopy and lifetime imaging, and can aid in optimizing future fluorescent ligand development.

Optotracing for selective fluorescence-based detection, visualization and quantification of live S. aureus in real-time.

Methods for bacterial detection are needed to advance the infection research and diagnostics. Based on conformation-sensitive fluorescent tracer molecules, optotracing was recently established for dynamic detection and visualization of structural amyloids and polysaccharides in the biofilm matrix of gram-negative bacteria. Here, we extend the use of optotracing for detection of gram-positive bacteria, focussing on the clinically relevant opportunistic human pathogen Staphylococcus aureus. We identify a donor-acceptor-donor-type optotracer, whose binding-induced fluorescence enables real-time detection, quantification, and visualization of S. aureus in monoculture and when mixed with gram-negative Salmonella Enteritidis. An algorithm-based automated high-throughput screen of 1920 S. aureus transposon mutants recognized the cell envelope as the binding target, which was corroborated by super-resolution microscopy of bacterial cells and spectroscopic analysis of purified cell wall components. The binding event was essentially governed by hydrophobic interactions, which permitted custom-designed tuning of the binding selectivity towards S. aureus versus Enterococcus faecalis by appropriate selection of buffer conditions. Collectively this work demonstrates optotracing as an enabling technology relevant for any field of basic and applied research, where visualization and detection of S. aureus is needed.

Synthesis and Characterization of Thiophene-based Donor-Acceptor-Donor Heptameric Ligands for Spectral Assignment of Polymorphic Amyloid-ß Deposits.

Protein deposits are associated with many devastating diseases and fluorescent ligands able to visualize these pathological entities are essential. Here, we report the synthesis of thiophene-based donor-acceptor-donor heptameric ligands that can be utilized for spectral assignment of distinct amyloid-ß (Aß) aggregates, one of the pathological hallmarks in Alzheimer's disease. The ability of the ligands to selectively distinguish Aß deposits was abolished when the chemical composition of the ligands was altered. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between aggregated proteinaceous species consisting of the same peptide or protein. In addition, such ligands might aid in interpreting the potential role of polymorphic Aß deposits in the pathogenesis of Alzheimer's disease.

Discriminating α-synuclein strains in Parkinson's disease and multiple system atrophy.

Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein, including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy1. Clinically, it is challenging to differentiate Parkinson's disease and multiple system atrophy, especially at the early stages of disease2. Aggregates of α-synuclein in distinct synucleinopathies have been proposed to represent different conformational strains of α-synuclein that can self-propagate and spread from cell to cell3-6. Protein misfolding cyclic amplification (PMCA) is a technique that has previously been used to detect α-synuclein aggregates in samples of cerebrospinal fluid with high sensitivity and specificity7,8. Here we show that the α-synuclein-PMCA assay can discriminate between samples of cerebrospinal fluid from patients diagnosed with Parkinson's disease and samples from patients with multiple system atrophy, with an overall sensitivity of 95.4%. We used a combination of biochemical, biophysical and biological methods to analyse the product of α-synuclein-PMCA, and found that the characteristics of the α-synuclein aggregates in the cerebrospinal fluid could be used to readily distinguish between Parkinson's disease and multiple system atrophy. We also found that the properties of aggregates that were amplified from the cerebrospinal fluid were similar to those of aggregates that were amplified from the brain. These findings suggest that α-synuclein aggregates that are associated with Parkinson's disease and multiple system atrophy correspond to different conformational strains of α-synuclein, which can be amplified and detected by α-synuclein-PMCA. Our results may help to improve our understanding of the mechanism of α-synuclein misfolding and the structures of the aggregates that are implicated in different synucleinopathies, and may also enable the development of a biochemical assay to discriminate between Parkinson's disease and multiple system atrophy.

In vivo detection of tau fibrils and amyloid ß aggregates with luminescent conjugated oligothiophenes and multiphoton microscopy.

The detection of amyloid beta deposits and neurofibrillary tangles, both hallmarks of Alzheimer's disease (AD), is key to understanding the mechanisms underlying these pathologies. Luminescent conjugated oligothiophenes (LCOs) enable fluorescence imaging of these protein aggregates. Using LCOs and multiphoton microscopy, individual tangles and amyloid beta deposits were labeled in vivo and imaged longitudinally in a mouse model of tauopathy and cerebral amyloidosis, respectively. Importantly, LCO HS-84, whose emission falls in the green region of the spectrum, allowed for the first time longitudinal imaging of tangle dynamics following a single intravenous injection. In addition, LCO HS-169, whose emission falls in the red region of the spectrum, successfully labeled amyloid beta deposits, allowing multiplexing with other reporters whose emission falls in the green region of the spectrum. In conclusion, this method can provide a new approach for longitudinal in vivo imaging using multiphoton microscopy of AD pathologies as well as other neurodegenerative diseases associated with protein aggregation in mouse models.

Synthesis of Thiophene-Based Optical Ligands That Selectively Detect Tau Pathology in Alzheimer's Disease.

The accumulation of protein aggregates is associated with many devastating neurodegenerative diseases and the development of molecular ligands able to detect these pathological hallmarks is essential. Here, the synthesis of thiophene based optical ligands, denoted bi-thiophene-vinyl-benzothiazoles (bTVBTs) that can be utilized for selective assignment of tau aggregates in brain tissue with Alzheimer's disease (AD) pathology is reported. The ability of the ligands to selectively distinguish tau deposits from the other AD associated pathological hallmark, senile plaques consisting of aggregated amyloid-ß (Aß) peptide, was reduced when the chemical composition of the ligands was altered, verifying that specific molecular interactions between the ligands and the aggregates are necessary for the selective detection of tau deposits. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between aggregated proteinaceous species consisting of different proteins. In addition, the bTVBT scaffold might be utilized to create powerful practical research tools for studying the underlying molecular events of tau aggregation and for creating novel agents for clinical imaging of tau pathology in AD.

A Palette of Fluorescent Thiophene-Based Ligands for the Identification of Protein Aggregates.

By replacing the central thiophene unit of an anionic pentameric oligothiophene with other heterocyclic moities, a palette of pentameric thiophene-based ligands with distinct fluorescent properties were synthesized. All ligands displayed superior selectivity towards recombinant amyloid fibrils as well as disease-associated protein aggregates in tissue sections.

Structure-based drug design identifies polythiophenes as antiprion compounds.

Prions cause transmissible spongiform encephalopathies for which no treatment exists. Prions consist of PrP(Sc), a misfolded and aggregated form of the cellular prion protein (PrP(C)). We explore the antiprion properties of luminescent conjugated polythiophenes (LCPs) that bind and stabilize ordered protein aggregates. By administering a library of structurally diverse LCPs to the brains of prion-infected mice via osmotic minipumps, we found that antiprion activity required a minimum of five thiophene rings bearing regularly spaced carboxyl side groups. Solid-state nuclear magnetic resonance analyses and molecular dynamics simulations revealed that anionic side chains interacted with complementary, regularly spaced cationic amyloid residues of model prions. These findings allowed us to extract structural rules governing the interaction between LCPs and protein aggregates, which we then used to design a new set of LCPs with optimized binding. The new set of LCPs showed robust prophylactic and therapeutic potency in prion-infected mice, with the lead compound extending survival by >80% and showing activity against both mouse and hamster prions as well as efficacy upon intraperitoneal administration into mice. These results demonstrate the feasibility of targeted chemical design of compounds that may be useful for treating diseases of aberrant protein aggregation such as prion disease.

Distinct Spacing Between Anionic Groups: An Essential Chemical Determinant for Achieving Thiophene-Based Ligands to Distinguish ß-Amyloid or Tau Polymorphic Aggregates.

The accumulation of protein aggregates is associated with many devastating neurodegenerative diseases and the existence of distinct aggregated morphotypes has been suggested to explain the heterogeneous phenotype reported for these diseases. Thus, the development of molecular probes able to distinguish such morphotypes is essential. We report an anionic tetrameric oligothiophene compound that can be utilized for spectral assignment of different morphotypes of ß-amyloid or tau aggregates present in transgenic mice at distinct ages. The ability of the ligand to spectrally distinguish between the aggregated morphotypes was reduced when the spacing between the anionic substituents along the conjugated thiophene backbone was altered, which verified that specific molecular interactions between the ligand and the protein aggregate are necessary to detect aggregate polymorphism. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between different morphotypes of protein aggregates.

Pentameric thiophene-based ligands that spectrally discriminate amyloid-ß and tau aggregates display distinct solvatochromism and viscosity-induced spectral shifts.

A wide range of neurodegenerative diseases are characterized by the deposition of multiple protein aggregates. Ligands for molecular characterization and discrimination of these pathological hallmarks are thus important for understanding their potential role in pathogenesis as well as for clinical diagnosis of the disease. In this regard, luminescent conjugated oligothiophenes (LCOs) have proven useful for spectral discrimination of amyloid-beta (Aß) and tau neurofibrillary tangles (NFTs), two of the pathological hallmarks associated with Alzheimer's disease. Herein, the solvatochromism of a library of anionic pentameric thiophene-based ligands, as well as their ability to spectrally discriminate Aß and tau aggregates, were investigated. Overall, the results from this study identified distinct solvatochromic and viscosity-dependent behavior of thiophene-based ligands that can be applied as indices to direct the chemical design of improved LCOs for spectral separation of Aß and tau aggregates in brain tissue sections. The results also suggest that the observed spectral transitions of the ligands are due to their ability to conform by induced fit to specific microenvironments within the binding interface of each particular protein aggregate. We foresee that these findings might aid in the chemical design of thiophene-based ligands that are increasingly selective for distinct disease-associated protein aggregates.