RESUMO
The amyloid ß (Aß) peptide has a central role in Alzheimer's disease (AD) pathology. The peptide length can vary between 37 and 49 amino acids, with Aß1-42 being considered the most disease-related length. However, Aß1-40 is also found in Aß plaques and has shown to form intertwined fibrils with Aß1-42. The peptides have previously also shown to form different fibril conformations, proposed to be related to disease phenotype. To conduct more representative in vitro experiments, it is vital to uncover the impact of different fibril conformations on neurons. Hence, we fibrillized different Aß1-40:42 ratios in concentrations of 100:0, 90:10, 75:25, 50:50, 25:75, 10:90 and 0:100 for either 24 h (early fibrils) or 7 days (aged fibrils). These were then characterized based on fibril width, LCO-staining and antibody-staining. We further challenged differentiated neuronal-like SH-SY5Y human cells with the different fibrils and measured Aß content, cytotoxicity and autophagy function at three different time-points: 3, 24, and 72 h. Our results revealed that both Aß1-40:42 ratio and fibril maturation affect conformation of fibrils. We further show the impact of these conformation changes on the affinity to commonly used Aß antibodies, primarily affecting Aß1-40 rich aggregates. In addition, we demonstrate uptake of the aggregates by neuronally differentiated human cells, where aggregates with higher Aß1-42 ratios generally caused higher cellular levels of Aß. These differences in Aß abundance did not cause changes in cytotoxicity nor in autophagy activation. Our results show the importance to consider conformational differences of Aß fibrils, as this can have fundamental impact on Aß antibody detection. Overall, these insights underline the need for further exploration of the impact of conformationally different fibrils and the need to reliably produce disease relevant Aß aggregates.
RESUMO
PURPOSE: To assess the analgesic and anxiolytic effects of virtual reality (VR) augmentation in patients undergoing peripherally inserted central catheter (PICC) placement or fine-needle aspiration thyroid biopsy. MATERIALS AND METHODS: This is a prospective, single-center randomized controlled trial with 107 patients enrolled. Patients were randomly assigned to receive standard of care (SOC) or SOC+VR during PICC or thyroid biopsy procedures. Pain and anxiety were individually measured using the visual analog scale (VAS) before and after the procedure. Vital signs including heart rate and systolic and diastolic blood pressure were recorded. One-way analysis of variance test and Games-Howell post hoc analysis were used to assess effect size and statistical significance between SOC and SOC+VR measures. RESULTS: The PICC cohort consisted of 59 patients (33 in SOC+VR and 26 in SOC), with a median age of 53.1 years (interquartile range [IQR], 38.3-62.7 years). The thyroid biopsy cohort consisted of 48 patients (26 in SOC+VR and 22 in SOC), with a median age of 60.1 years (IQR, 49.0-67.2 years). One-way analysis of individuals undergoing thyroid biopsies with adjunctive VR revealed an effect size of -1.74 points (SE ± 0.71; P = .018) on VAS pain scale when compared with SOC. Analysis of individuals undergoing PICC placements revealed an effect size of -1.60 points (SE ± 0.81; P = .053) on VAS anxiety when compared with SOC. CONCLUSIONS: VR as a nonpharmacologic adjunct reduced some procedure-related pain and anxiety without increasing the procedural duration.
Assuntos
Ansiedade , Cateterismo Periférico , Medição da Dor , Humanos , Pessoa de Meia-Idade , Masculino , Feminino , Estudos Prospectivos , Projetos Piloto , Ansiedade/prevenção & controle , Adulto , Idoso , Cateterismo Periférico/efeitos adversos , Resultado do Tratamento , Manejo da Dor , Dor Processual/etiologia , Dor Processual/prevenção & controle , Dor Processual/diagnóstico , Cateterismo Venoso Central/efeitos adversos , Realidade Virtual , Terapia de Exposição à Realidade Virtual , Radiografia IntervencionistaRESUMO
CBD-2115 was selected from a library of 148 compounds based on a pyridinyl-indole scaffold as a first-in-class 4R-tau radiotracer. In vitro binding assays showed [3H]CBD-2115 had a KD value of 6.9 nM and a nominal Bmax of 500 nM in 4R-tau expressing P301L transgenic mouse tissue. In binding assays with human brain tissue homogenates, [3H]CBD-2115 has a higher affinity (4.9 nM) for progressive supranuclear palsy specific 4R-tau deposits than [3H]flortaucipir (45 nM) or [3H]MK-6240 (>50 nM). [18F]CBD-2115 was reliably synthesized (3-11% radiochemical yield with molar activity of 27-111 GBq/µmol and >97% radiochemical purity). Dynamic PET imaging was conducted in mice, rats, and nonhuman primates, and all species showed initial brain uptake of 0.5-0.65 standardized uptake value with fast clearance from normal tissues. [3H]CBD-2115 could be a useful lead radioligand for further research in 4R-tauopathies, and PET radiotracer development will focus on improving brain uptake and binding affinity.
Assuntos
Tauopatias , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Camundongos , Tomografia por Emissão de Pósitrons , Radioquímica , Compostos Radiofarmacêuticos , Ratos , Proteínas tau/metabolismoRESUMO
Microtubule Associated Protein Tau (MAPT) forms proteopathic aggregates in several diseases. The G273R tau mutation, located in the first repeat region, was found by exome sequencing in a patient who presented with dementia and parkinsonism. We herein return to pathological examination which demonstrated tau immunoreactivity in neurons and glia consistent of mixed progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) features. To rationalize the pathological findings, we used molecular biophysics to characterize the mutation in more detail in vitro and in Drosophila. The G273R mutation increases the aggregation propensity of 4-repeat (4R) tau and alters the tau binding affinity towards microtubules (MTs) and F-actin. Tau aggregates in PSP and CBD are predominantly 4R tau. Our data suggest that the G273R mutation induces a shift in pool of 4R tau by lower F-actin affinity, alters the conformation of MT bound 4R tau, while increasing chaperoning of 3R tau by binding stronger to F-actin. The mutation augmented fibrillation of 4R tau initiation in vitro and in glial cells in Drosophila and showed preferential seeding of 4R tau in vitro suggestively causing a late onset 4R tauopathy reminiscent of PSP and CBD.
Assuntos
Encéfalo/patologia , Neurônios/metabolismo , Paralisia Supranuclear Progressiva/metabolismo , Tauopatias/patologia , Animais , Doenças dos Gânglios da Base/metabolismo , Encéfalo/metabolismo , Drosophila , Mutação/genética , Neuroglia/metabolismoAssuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Agregados Proteicos , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Amiloidose , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Drosophila melanogaster , Humanos , Camundongos , Fragmentos de Peptídeos/genéticaRESUMO
A fluorescent bis-styryl-benzothiadiazole (BTD) with carboxylic acid functional groups (X-34/Congo red analogue) showed lower binding affinity toward Aß1-42 and Aß1-40 fibrils than its neutral analogue. Hence, variable patterns of neutral OH-substituted bis-styryl-BTDs were generated. All bis-styryl-BTDs showed higher binding affinity to Aß1-42 fibrils than to Aß1-40 fibrils. The para-OH on the phenyl rings was beneficial for binding affinity while a meta-OH decreased the affinity. Differential staining of transgenic mouse Aß amyloid plaque cores compared to peripheral coronas using neutral compared to anionic bis-styryl ligands indicate differential recognition of amyloid polymorphs. Hyperspectral imaging of transgenic mouse Aß plaque stained with uncharged para-hydroxyl substituted bis-styryl-BTD implicated differences in binding site polarity of polymorphic amyloid plaque. Most properties of the corresponding bis-styryl-BTD were retained with a rigid alkyne linker rendering a probe insensitive to cis-trans isomerization. These new BTD-based ligands are promising probes for spectral imaging of different Aß fibril polymorphs.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Corantes Fluorescentes/farmacologia , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/metabolismo , Estirenos/farmacologia , Tiadiazóis/farmacologia , Animais , Feminino , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Ligantes , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência , Ligação Proteica , Estirenos/síntese química , Estirenos/metabolismo , Tiadiazóis/síntese química , Tiadiazóis/metabolismoRESUMO
Two analogues to the fluorescent amyloid probe 2,5-bis(4'-hydroxy-3'-carboxy-styryl)benzene (X-34) were synthesized based on the trans-stilbene pyrene scaffold (Py1SA and Py2SA). The compounds show strikingly different emission spectra when bound to preformed Aß1-42 fibrils. This remarkable emission difference is retained when bound to amyloid fibrils of four distinct proteins, suggesting a common binding configuration for each molecule. Density functional theory calculations show that Py1SA is twisted, while Py2SA is more planar. Still, an analysis of the highest occupied molecular orbitals (HOMOs) and lowest unoccupied molecular orbitals (LUMOs) of the two compounds indicates that the degree of electronic coupling between the pyrene and salicylic acid (SA) moieties is larger in Py1SA than in Py2SA. Excited state intramolecular proton transfer (ESIPT) coupled-charge transfer (ICT) was observed for the anionic form in polar solvents. We conclude that ICT properties of trans-stilbene derivatives can be utilized for amyloid probe design with large changes in emission spectra and decay times from analogous chemical structures depending on the detailed physical nature of the binding site.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Prótons , Pirenos/química , Salicilatos/química , Estilbenos/química , Teoria da Densidade Funcional , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/efeitos da radiação , Luz , Modelos Químicos , Estrutura Molecular , Multimerização Proteica , Pirenos/síntese química , Pirenos/efeitos da radiação , Salicilatos/síntese química , Salicilatos/efeitos da radiação , Estilbenos/síntese química , Estilbenos/efeitos da radiaçãoRESUMO
Protein misfolding, aggregation, and amyloid formation is involved in a large number of diseases. Recombinantly expressed proteins to study the amyloid fibril formation process are important for mechanistic studies. We here report protocols for production, purification, and fibrillation of three different proteins commonly found in cerebral amyloid; Aß and Tau found in Alzheimer's disease, Chronic traumatic brain injury, Corticobasal degeneration, and Progressive Supranuclear Palsy and human prion protein found in Creutzfeldt-Jakob's disease. The three protocols have in common that the protein is in a pH-neutral phosphate saline buffer during fibrillation to mimic their endogenous near physiological environment.
Assuntos
Peptídeos beta-Amiloides/isolamento & purificação , Doenças Neurodegenerativas/metabolismo , Proteínas Priônicas/isolamento & purificação , Proteínas tau/isolamento & purificação , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Cromatografia por Troca Iônica , Síndrome de Creutzfeldt-Jakob/metabolismo , Humanos , Proteínas Priônicas/química , Dobramento de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas tau/químicaRESUMO
The basis for selective vulnerability of certain cell types for misfolded proteins (MPs) in neurodegenerative diseases is largely unknown. This knowledge is crucial for understanding disease progression in relation to MPs spreading in the CNS. We assessed this issue in Drosophila by cell-specific expression of human Aß1-42 associated with Alzheimer's disease. Expression of Aß1-42 in various neurons resulted in concentration-dependent severe neurodegenerative phenotypes, and intraneuronal ring-tangle-like aggregates with immature fibril properties when analyzed by aggregate-specific ligands. Unexpectedly, expression of Aß1-42 from a pan-glial driver produced a mild phenotype despite massive brain load of Aß1-42 aggregates, even higher than in the strongest neuronal driver. Glial cells formed more mature fibrous aggregates, morphologically distinct from aggregates found in neurons, and was mainly extracellular. Our findings implicate that Aß1-42 cytotoxicity is both cell and aggregate morphotype dependent.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Drosophila/metabolismo , Neuroglia/patologia , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Agregação Patológica de Proteínas/patologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Animais , Modelos Animais de Doenças , Humanos , Neuroglia/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Agregação Patológica de Proteínas/metabolismoRESUMO
We revisited the Congo red analogue 2,5-bis(4'-hydroxy-3'-carboxy-styryl)benzene (X-34) to develop this highly fluorescent amyloid dye for imaging Alzheimer's disease (AD) pathology comprising Aß and Tau fibrils. A selection of ligands with distinct optical properties were synthesized by replacing the central benzene unit of X-34, with other heterocyclic moieties. Full photophysical characterization was performed, including recording absorbance and fluorescence spectra, Stokes shift, quantum yield and fluorescence lifetimes. All ligands displayed high affinity towards recombinant amyloid fibrils of Aß1-42 (13-300â nm Kd ) and Tau (16-200â nm Kd ) as well as selectivity towards the corresponding disease-associated protein aggregates in AD tissue. We observed that these ligands efficiently displaced X-34, but not Pittsburgh compound B (PiB) from recombinant Aß1-42 amyloid fibrils, arguing for retained targeting of the Congo red type binding site. We foresee that the X-34 scaffold offers the possibility to develop novel high-affinity ligands for Aß pathology found in human AD brain in a different mode compared with PiB, potentially recognizing different polymorphs of Aß fibrils.
Assuntos
Alcenos/química , Peptídeos beta-Amiloides/química , Amiloide/química , Amiloide/metabolismo , Compostos de Anilina/química , Benzoatos/química , Corantes Fluorescentes/química , Tiazóis/química , Proteínas tau/química , Peptídeos beta-Amiloides/metabolismo , HumanosRESUMO
It was previously reported that two naphthyl-based trans-stilbene probes, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (1) and (E)-4-(2-(naphthalen-2-yl)vinyl)benzene-1,2-diol (3), can bind to both native transthyretin (TTR) and misfolded protofibrillar TTR at physiological concentrations, displaying distinct emission maxima bound to the different conformational states (>100 nm difference). To further explore this amyloid probe scaffold to obtain extended fluorescence lifetimes, two new analogues with expanded aromatic ring systems (anthracene and pyrene), (E)-4-(2-(anthracen-2-yl)vinyl)benzene-1,2-diol (4) and (E)-4-(2-(pyren-2-yl)vinyl)benzene-1,2-diol (5), were synthesized employing the palladium-catalyzed Mizoroki-Heck reaction. (E)-4-Styrylbenzene-1,2-diol (2), 3, 4, and 5 were investigated with respect to their photophysical properties in methanol and when bound to insulin, lysozyme, and Aß1-42 fibrils, including time-resolved fluorescence measurements. In conclusion, 4 and 5 can bind to both native and fibrillar TTR, becoming highly fluorescent. Compounds 2-5 bind specifically to insulin, lysozyme, and Aß1-42 fibrils with an apparent fluorescence intensity increase and moderate binding affinities. The average fluorescence lifetimes of the probes bound to Aß1-42 fibrils are 1.3 ns (2), 1.5 ns (3), 5.7 ns (4), and 29.8 ns (5). In summary, the variable aromatic moieties of the para-positioned trans-stilbenoid vinyl-benzene-1,2-diol with benzene, naphthalene, anthracene, and pyrene showed that the extended conjugated systems retained the amyloid targeting properties of the probes. Furthermore, both the anthracene and pyrene moieties extensively enhanced the fluorescence intensity and prolonged lifetimes. These attractive probe properties should improve amyloid detection and characterization by fluorescence-based techniques.