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.