In vitro metabolism study of ADB-P-5Br-INACA and ADB-4en-P-5Br-INACA using human hepatocytes, liver microsomes, and in-house synthesized references.

Synthetic cannabinoids (SCs) remain a major public health concern, as they continuously are linked to severe intoxications and drug-related deaths worldwide. As new SCs continue to emerge on the illicit drug market, an understanding of SC metabolism is needed to identify formed metabolites that may serve as biomarkers in forensic toxicology screening and for understanding the pharmacokinetics of the drugs. In this work, the metabolism of ADB-4en-P-5Br-INACA and ADB-P-5Br-INACA ((S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-5-bromo-1-(pent-4-en-1-yl)-1H-indazole-3-carboxamide, (S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-5-bromo-1-pentyl-1H-indazole-3-carboxamide respectively) were investigated using human hepatocytes in vitro and in-house synthesized references. Both SCs were incubated with pooled human hepatocytes over 3 h, with the aim to identify unique and abundant metabolites using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In total nine metabolites were identified for ADB-4en-P-5Br-INACA and 10 metabolites for ADB-P-5Br-INACA. The observed biotransformations included dihydrodiol formation, terminal amide hydrolysis, hydroxylation, dehydrogenation, carbonyl formation, glucuronidation, and combinations thereof. The major metabolites were confirmed by in-house synthesized references. Recommended biomarkers for ADB-P-5Br-INACA and ADB-4en-P-5Br-INACA are the terminal hydroxy and dihydrodiol metabolite respectively.

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.

A Novel Drosophila Model of Alzheimer's Disease to Study Aß Proteotoxicity in the Digestive Tract.

Amyloid-ß (Aß) proteotoxicity is associated with Alzheimer's disease (AD) and is caused by protein aggregation, resulting in neuronal damage in the brain. In the search for novel treatments, Drosophila melanogaster has been extensively used to screen for anti-Aß proteotoxic agents in studies where toxic Aß peptides are expressed in the fly brain. Since drug molecules often are administered orally there is a risk that they fail to reach the brain, due to their inability to cross the brain barrier. To circumvent this problem, we have designed a novel Drosophila model that expresses the Aß peptides in the digestive tract. In addition, a built-in apoptotic sensor provides a fluorescent signal from the green fluorescent protein as a response to caspase activity. We found that expressing different variants of Aß1-42 resulted in proteotoxic phenotypes such as reduced longevity, aggregate deposition, and the presence of apoptotic cells. Taken together, this gut-based Aß-expressing fly model can be used to study the mechanisms behind Aß proteotoxicity and to identify different substances that can modify Aß proteotoxicity.

Mutation ∆K281 in MAPT causes Pick's disease.

Two siblings with deletion mutation ∆K281 in MAPT developed frontotemporal dementia. At autopsy, numerous inclusions of hyperphosphorylated 3R Tau were present in neurons and glial cells of neocortex and some subcortical regions, including hippocampus, caudate/putamen and globus pallidus. The inclusions were argyrophilic with Bodian silver, but not with Gallyas-Braak silver. They were not labelled by an antibody specific for tau phosphorylated at S262 and/or S356. The inclusions were stained by luminescent conjugated oligothiophene HS-84, but not by bTVBT4. Electron cryo-microscopy revealed that the core of tau filaments was made of residues K254-F378 of 3R Tau and was indistinguishable from that of Pick's disease. We conclude that MAPT mutation ∆K281 causes Pick's disease.

Thiophene-Based Ligands: Design, Synthesis and Their Utilization for Optical Assignment of Polymorphic-Disease-Associated Protein Aggregates.

The development of ligands for detecting protein aggregates is of great interest, as these aggregated proteinaceous species are the pathological hallmarks of several devastating diseases, including Alzheimer's disease. In this regard, thiophene-based ligands have emerged as powerful tools for fluorescent assessment of these pathological entities. The intrinsic conformationally sensitive photophysical properties of poly- and oligothiophenes have allowed optical assignment of disease-associated protein aggregates in tissue sections, as well as real-time in vivo imaging of protein deposits. Herein, we recount the chemical evolution of different generations of thiophene-based ligands, and exemplify their use for the optical distinction of polymorphic protein aggregates. Furthermore, the chemical determinants for achieving a superior fluorescent thiophene-based ligand, as well as the next generation of thiophene-based ligands targeting distinct aggregated species are described. Finally, the directions for future research into the chemical design of thiophene-based ligands that can aid in resolving the scientific challenges around protein aggregation diseases are discussed.

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.

Host oligodendrogliopathy and α-synuclein strains dictate disease severity in multiple system atrophy.

Multiple system atrophy is a progressive neurodegenerative disease with prominent autonomic and motor features. During early stages, different subtypes of the disease are distinguished by their predominant parkinsonian or cerebellar symptoms, reflecting its heterogeneous nature. The pathognomonic feature of multiple system atrophy is the presence of α-synuclein (αSyn) protein deposits in oligodendroglial cells. αSyn can assemble in specific cellular or disease environments and form αSyn strains with unique structural features, but the ability of αSyn strains to propagate in oligodendrocytes remains elusive. Recently, it was shown that αSyn strains with related conformations exist in the brains of patients. Here, we investigated whether different αSyn strains can influence multiple system atrophy progression in a strain-dependent manner. To this aim, we injected two recombinant αSyn strains (fibrils and ribbons) in multiple system atrophy transgenic mice and found that they determined disease severity in multiple system atrophy via host-restricted and cell-specific pathology in vivo. αSyn strains significantly impact disease progression in a strain-dependent way via oligodendroglial, neurotoxic and immune-related mechanisms. Neurodegeneration and brain atrophy were accompanied by unique microglial and astroglial responses and the recruitment of central and peripheral immune cells. The differential activation of microglial cells correlated with the structural features of αSyn strains both in vitro and in vivo. Spectral analysis showed that ribbons propagated oligodendroglial inclusions that were structurally distinct from those of fibrils, with resemblance to oligodendroglial inclusions, in the brains of patients with multiple system atrophy. This study, therefore, shows that the multiple system atrophy phenotype is governed by both the nature of the αSyn strain and the host environment and that by injecting αSyn strains into an animal model of the disease, a more comprehensive phenotype can be established.

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.

Proteophenes - Amino Acid Functionalized Thiophene-based Fluorescent Ligands for Visualization of Protein Deposits in Tissue Sections with Alzheimer's Disease Pathology.

Protein deposits composed of specific proteins or peptides are associated with several neurodegenerative diseases and fluorescent ligands able to detect these pathological hallmarks are vital. Here, we report the synthesis of a class of thiophene-based ligands, denoted proteophenes, with different amino acid side-chain functionalities along the conjugated backbone, which display selectivity towards specific disease-associated protein aggregates in tissue sections with Alzheimer's disease (AD) pathology. The selectivity of the ligands towards AD associated pathological hallmarks, such as aggregates of the amyloid-ß (Aß) peptide or tau filamentous inclusions, was highly dependent on the chemical nature of the amino acid functionality, as well as on the location of the functionality along the pentameric thiophene backbone. Finally, the concept of synthesizing donor-acceptor-donor proteophenes with distinct photophysical properties was shown. Our findings provide the structural and functional basis for the development of new thiophene-based ligands that can be utilized for optical assignment of different aggregated proteinaceous species in tissue sections.

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.

Tau Protein Binding Modes in Alzheimer's Disease for Cationic Luminescent Ligands.

The bi-thiophene-vinylene-benzothiazole (bTVBT4) ligand developed for Alzheimer's disease (AD)-specific detection of amyloid tau has been studied by a combination of several theoretical methods and experimental spectroscopies. With reference to the cryo-EM tau structure of the tau protofilament ( Nature 2017, 547, 185), a periodic model system of the fibril was created, and the interactions between this fibril and bTVBT4 were studied with nonbiased molecular dynamics simulations. Several binding sites and binding modes were identified and analyzed, and the results for the most prevailing fibril site and ligand modes are presented. A key validation of the simulation work is provided by the favorable comparison of the theoretical and experimental absorption spectra of bTVBT4 in solution and bound to the protein. It is conclusively shown that the ligand-protein binding occurs at the hydrophobic pocket defined by the residues Ile360, Thr361, and His362. This binding site is not accessible in the Pick's disease (PiD) fold, and fluorescence imaging of bTVBT4-stained brain tissue samples from patients diagnosed with AD and PiD provides strong support for the proposed tau binding site.

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.

Accumulation of alpha-synuclein within the liver, potential role in the clearance of brain pathology associated with Parkinson's disease.

Alpha-synuclein (α-syn) aggregation is the hallmark pathological lesion in brains of patients with Parkinson's disease (PD) and related neurological disorders characterized as synucleinopathies. Accumulating evidence now indicates that α-syn deposition is also present within the gut and other peripheral organs outside the central nervous system (CNS). In the current study, we demonstrate for the first time that α-syn pathology also accumulates within the liver, the main organ responsible for substance clearance and detoxification. We further demonstrate that cultured human hepatocytes readily internalize oligomeric α-syn assemblies mediated, at least in part, by the gap junction protein connexin-32 (Cx32). Moreover, we identified a time-dependent accumulation of α-syn within the liver of three different transgenic (tg) mouse models expressing human α-syn under CNS-specific promoters, despite the lack of α-syn mRNA expression within the liver. Such a brain-to-liver transmission route could be further corroborated by detection of α-syn pathology within the liver of wild type mice one month after a single striatal α-syn injection. In contrast to the synucleinopathy models, aged mice modeling AD rarely show any amyloid-beta (Aß) deposition within the liver. In human post-mortem liver tissue, we identified cases with neuropathologically confirmed α-syn pathology containing α-syn within hepatocellular structures to a higher degree (75%) than control subjects without α-syn accumulation in the brain (57%). Our results reveal that α-syn accumulates within the liver and may be derived from the brain or other peripheral sources. Collectively, our findings indicate that the liver may play a role in the clearance and detoxification of pathological proteins in PD and related synucleinopathies.

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.

Luminescent conjugated oligothiophenes distinguish between α-synuclein assemblies of Parkinson's disease and multiple system atrophy.

Synucleinopathies [Parkinson's disease with or without dementia, dementia with Lewy bodies and multiple system atrophy] are neurodegenerative diseases that are defined by the presence of filamentous α-synuclein inclusions. We investigated the ability of luminescent conjugated oligothiophenes to stain the inclusions of Parkinson's disease and multiple system atrophy. They stained the Lewy pathology of Parkinson's disease and the glial cytoplasmic inclusions of multiple system atrophy. Spectral analysis of HS-68-stained inclusions showed a red shift in multiple system atrophy, but the difference with Parkinson's disease was not significant. However, when inclusions were double-labelled for HS-68 and an antibody specific for α-synuclein phosphorylated at S129, they could be distinguished based on colour shifts with blue designated for Parkinson's disease and red for multiple system atrophy. The inclusions of Parkinson's disease and multiple system atrophy could also be distinguished using fluorescence lifetime imaging. These findings are consistent with the presence of distinct conformers of assembled α-synuclein in Parkinson's disease and multiple system atrophy.

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.

The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice.

Identification of fluorescent dyes that label the filamentous protein aggregates characteristic of neurodegenerative disease, such as ß-amyloid and tau in Alzheimer's disease, in a live cell culture system has previously been a major hurdle. Here we show that pentameric formyl thiophene acetic acid (pFTAA) fulfills this function in living neurons cultured from adult P301S tau transgenic mice. Injection of pFTAA into 5-month-old P301S tau mice detected cortical and DRG neurons immunoreactive for AT100, an antibody that identifies solely filamentous tau, or MC1, an antibody that identifies a conformational change in tau that is commensurate with neurofibrillary tangle formation in Alzheimer's disease brains. In fixed cultures of dorsal root ganglion (DRG) neurons, pFTAA binding, which also identified AT100 or MC1+ve neurons, followed a single, saturable binding curve with a half saturation constant of 0.14 µM, the first reported measurement of a binding affinity of a beta-sheet reactive dye to primary neurons harboring filamentous tau. Treatment with formic acid, which solubilizes filamentous tau, extracted pFTAA, and prevented the re-binding of pFTAA and MC1 without perturbing expression of soluble tau, detected using an anti-human tau (HT7) antibody. In live cultures, pFTAA only identified DRG neurons that, after fixation, were AT100/MC1+ve, confirming that these forms of tau pre-exist in live neurons. The utility of pFTAA to discriminate between living neurons containing filamentous tau from other neurons is demonstrated by showing that more pFTAA+ve neurons die than pFTAA-ve neurons over 25 days. Since pFTAA identifies fibrillar tau and other misfolded proteins in living neurons in culture and in animal models of several neurodegenerative diseases, as well as in human brains, it will have considerable application in sorting out disease mechanisms and in identifying disease-modifying drugs that will ultimately help establish the mechanisms of neurodegeneration in human neurodegenerative diseases.

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.

The structural basis for optimal performance of oligothiophene-based fluorescent amyloid ligands: conformational flexibility is essential for spectral assignment of a diversity of protein aggregates.

Protein misfolding diseases are characterized by deposition of protein aggregates, and optical ligands for molecular characterization of these disease-associated structures are important for understanding their potential role in the pathogenesis of the disease. Luminescent conjugated oligothiophenes (LCOs) have proven useful for optical identification of a broader subset of disease-associated protein aggregates than conventional ligands, such as thioflavin T and Congo red. Herein, the molecular requirements for achieving LCOs able to detect nonthioflavinophilic Aß aggregates or non-congophilic prion aggregates, as well as spectrally discriminate Aß and tau aggregates, were investigated. An anionic pentameric LCO was subjected to chemical engineering by: 1) replacing thiophene units with selenophene or phenylene moieties, or 2) alternating the anionic substituents along the thiophene backbone. In addition, two asymmetric tetrameric ligands were generated. Overall, the results from this study identified conformational freedom and extended conjugation of the conjugated backbone as crucial determinants for obtaining superior thiophene-based optical ligands for sensitive detection and spectral assignment of disease-associated protein aggregates.

Luminescent conjugated oligothiophenes for sensitive fluorescent assignment of protein inclusion bodies.

Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands.