Alzheimer's Deposits Research Articles

Distinct Chemical Determinants are Essential for Achieving Ligands for Superior Optical Detection of Specific Amyloid-β Deposits in Alzheimer's Disease.

Aggregated forms of different proteins are common hallmarks for several neurodegenerative diseases, including Alzheimer's disease, and ligands that selectively detect specific protein aggregates are vital. Herein, we investigate the molecular requirements of thiophene-vinyl-benzothiazole based ligands to detect a specific type of Aβ deposits found in individuals with dominantly inherited Alzheimer's disease caused by the Arctic APP E693G mutation. The staining of these Aβ deposits was alternated when switching the terminal heterocyclic moiety attached to the thiophene-vinyl-benzothiazole scaffold. The most prevalent staining was observed for ligands having a terminal 3-methyl-1H-indazole moiety or a terminal 1,2-dimethoxybenzene moiety, verifying that specific molecular interactions between these ligands and the aggregates were necessary. The synthesis of additional thiophene-vinyl-benzothiazole ligands aided in pinpointing additional crucial chemical determinants, such as positioning of nitrogen atoms and methyl substituents, for achieving optimal staining of Aβ aggregates. When combining the optimized thiophene-vinyl-benzothiazole based ligands with a conventional ligand, CN-PiB, distinct staining patterns were observed for sporadic Alzheimer's disease versus dominantly inherited Alzheimer's disease caused by the Arctic APP E693G mutation. Our findings provide chemical insights for developing novel ligands that allow for a more precise assignment of Aβ deposits, and might also aid in creating novel agents for clinical imaging of distinct Aβ aggregates in AD.

Probing the microstructure of pathological protein deposits in Alzheimer’s disease

Probing the microstructure of pathological protein deposits in Alzheimer’s disease

A Novel Near-Infrared Fluorescence Probe THK-565 Enables In Vivo Detection of Amyloid Deposits in Alzheimer’s Disease Mouse Model

PurposeNoninvasive imaging of protein aggregates in the brain is critical for the early diagnosis, disease monitoring, and evaluation of the effectiveness of novel therapies for Alzheimer’s disease (AD). Near-infrared fluorescence (NIRF) imaging with specific probes is a promising technique for the in vivo detection of protein deposits without radiation exposure. Comprehensive screening of fluorescent compounds identified a novel compound, THK-565, for the in vivo imaging of amyloid-β (Aβ) deposits in the mouse brain. This study assessed whether THK-565 could detect amyloid-β deposits in vivo in the AD mouse model.ProceduresThe fluorescent properties of THK-565 were evaluated in the presence and absence of Aβ fibrils. APP knock-in (APP-KI) mice were used as an animal model of AD. In vivo NIRF images were acquired after the intravenous administration of THK-565 and THK-265 in mice. The binding selectivity of THK-565 to Aβ was evaluated using brain slices obtained from these mouse models.ResultsThe fluorescence intensity of the THK-565 solution substantially increased by mixing with Aβ fibrils. The maximum emission wavelength of the complex of THK-565 and Aβ fibrils was 704 nm, which was within the optical window range. THK-565 selectively bound to amyloid deposits in brain sections of APP-KI mice After the intravenous administration of THK-565, the fluorescence signal in the head of APP-KI mice was significantly higher than that of wild-type mice and higher than that after administration of THK-265. Ex vivo analysis confirmed that the THK-565 signal corresponded to Aβ immunostaining in the brain sections of these mice.ConclusionsA novel NIRF probe, THK-565, enabled the in vivo detection of Aβ deposits in the brains of the AD mouse model, suggesting that NIRF imaging with THK-565 could non-invasively assess disease-specific pathology in AD.

Long-term effects of immunotherapy with a brain penetrating Aβ antibody in a mouse model of Alzheimer’s disease

BackgroundBrain-directed immunotherapy is a promising strategy to target amyloid-β (Aβ) deposits in Alzheimer’s disease (AD). In the present study, we compared the therapeutic efficacy of the Aβ protofibril targeting antibody RmAb158 with its bispecific variant RmAb158-scFv8D3, which enters the brain by transferrin receptor-mediated transcytosis.MethodsAppNL−G−F knock-in mice received RmAb158, RmAb158-scFv8D3, or PBS in three treatment regimens. First, to assess the acute therapeutic effect, a single antibody dose was given to 5 months old AppNL−G−F mice, with evaluation after 3 days. Second, to assess the antibodies’ ability to halt the progression of Aβ pathology, 3 months old AppNL−G−F mice received three doses during a week, with evaluation after 2 months. Reduction of RmAb158-scFv8D3 immunogenicity was explored by introducing mutations in the antibody or by depletion of CD4+ T cells. Third, to study the effects of chronic treatment, 7-month-old AppNL−G−F mice were CD4+ T cell depleted and treated with weekly antibody injections for 8 weeks, including a final diagnostic dose of [125I]RmAb158-scFv8D3, to determine its brain uptake ex vivo. Soluble Aβ aggregates and total Aβ42 were quantified with ELISA and immunostaining.ResultsNeither RmAb158-scFv8D3 nor RmAb158 reduced soluble Aβ protofibrils or insoluble Aβ1-42 after a single injection treatment. After three successive injections, Aβ1-42 was reduced in mice treated with RmAb158, with a similar trend in RmAb158-scFv8D3-treated mice. Bispecific antibody immunogenicity was somewhat reduced by directed mutations, but CD4+ T cell depletion was used for long-term therapy. CD4+ T cell-depleted mice, chronically treated with RmAb158-scFv8D3, showed a dose-dependent increase in blood concentration of the diagnostic [125I]RmAb158-scFv8D3, while concentration was low in plasma and brain. Chronic treatment did not affect soluble Aβ aggregates, but a reduction in total Aβ42 was seen in the cortex of mice treated with both antibodies.ConclusionsBoth RmAb158 and its bispecific variant RmAb158-scFv8D3 achieved positive effects of long-term treatment. Despite its ability to efficiently enter the brain, the benefit of using the bispecific antibody in chronic treatment was limited by its reduced plasma exposure, which may be a result of interactions with TfR or the immune system. Future research will focus in new antibody formats to further improve Aβ immunotherapy.

High-Affinity Fluorescent Probes for the Detection of Soluble and Insoluble Aβ Deposits in Alzheimer's Disease.

The overproduction and deposition of the amyloid-β (Aβ) aggregates are accountable for the genesis and development of the neurologic disorder Alzheimer's disease (AD). Effective medications and detection agents for AD are still deficient. General challenges for the diagnosis of Aβ aggregates in the AD brain are (i) crossing the blood-brain barrier (BBB) and (ii) selectivity to Aβ species with (iii) emission maxima in the 500-750 nm region. Thioflavin-T (ThT) is the most used fluorescent probe for imaging Aβ fibril aggregates. However, because of the poor BBB crossing (log P = -0.14) and short emission wavelength (482 nm) after binding with Aβ fibrils, ThT can be limited to in vitro use only. Herein, we have developed Aβ deposit-recognizing fluorescent probes (ARs) with a D-π-A architecture and a longer emission wavelength after binding with Aβ species. Among the newly designed probes, AR-14 showed an admirable fluorescence emission (>600 nm) change after binding with soluble Aβ oligomers (2.3-fold) and insoluble Aβ fibril aggregates (4.5-fold) with high affinities Kd = 24.25 ± 4.10 nM; Ka = (4.123 ± 0.69) × 107 M-1 for fibrils; Kd = 32.58 ± 4.89 nM; and Ka = (3.069 ± 0.46) × 107 M-1 for oligomers with high quantum yield, molecular weight of <500 Da, reasonable log P = 1.77, stability in serum, and nontoxicity, and it can cross the BBB efficiently. The binding affinity of AR-14 toward Aβ species is proved by fluorescence binding studies and fluorescent staining of 18-month-old triple-transgenic (3xTg) mouse brain sections. In summary, the fluorescent probe AR-14 is efficient and has an admirable quality for the detection of soluble and insoluble Aβ deposits in vitro and in vivo.

Structures of brain-derived 42-residue amyloid-β fibril polymorphs with unusual molecular conformations and intermolecular interactions

Fibrils formed by the 42-residue amyloid-β peptide (Aβ42), a main component of amyloid deposits in Alzheimer's disease (AD), are known to be polymorphic, i.e., to contain multiple possible molecular structures. Previous studies of Aβ42 fibrils, including fibrils prepared entirely invitro or extracted from brain tissue and using solid-state NMR (ssNMR) or cryogenic electron microscopy (cryo-EM) methods, have found polymorphs with differences in amino acid sidechain orientations, lengths of structurally ordered segments, and contacts between cross-β subunit pairs within a single filament. Despite these differences, Aβ42 molecules adopt a common S-shaped conformation in all previously described high-resolution Aβ42 fibril structures. Here we report two cryo-EM-based structures of Aβ42 fibrils that are qualitatively different, in samples derived from AD brain tissue by seeded growth. In type A fibrils, residues 12 to 42 adopt a ν-shaped conformation, with both intra-subunit and intersubunit hydrophobic contacts to form a compact core. In type B fibrils, residues 2 to 42 adopt an υ-shaped conformation, with only intersubunit contacts and internal pores. Type A and type B fibrils have opposite helical handedness. Cryo-EM density maps and molecular dynamics simulations indicate intersubunit K16-A42 salt bridges in type B fibrils and partially occupied K28-A42 salt bridges in type A fibrils. The coexistence of two predominant polymorphs, with differences in N-terminal dynamics, is supported by ssNMR data, as is faithful propagation of structures from first-generation to second-generation brain-seeded Aβ42 fibril samples. These results demonstrate that Aβ42 fibrils can exhibit a greater range of structural variations than seen in previous studies.

Microglia – An innocent bystander?

AbstractUnder normal conditions, microglia act as neuropathology sensors and are neuroprotective.Microglia are spatially organized features as mosaic in the retina; distributed in the plexiform layers of the retina and in the retinal nerve fibre layer‐ganglion cell layer, in order to control the environment and detect changes that can cause neuronal damage. Microglial cells may undergo two different types of activation in response to injuries, being an important player in maintaining retinal balance to a stressful situation. The microglia phenotype called M2‐like, secrete anti‐inflammatory mediators and neurotrophic factors that act as neuroprotectors, while the M1‐like generates a massive inflammatory response, being neurotoxic. When a neurodegenerative process occurs, such as that caused by protein deposits in Alzheimer's Disease (AD), the microglia responds changing its phenotype, which will cause changes in its shape, promoting migration to the area of damage, changing the arrangement in the tissue that gives rise to cells that converge or form barriers, isolating damaged tissue or phagocytizing toxic aggregate proteins and cellular debris.The migratory capacity of the microglia gives it the property of being able to move in its own layer and even between layers, remodelling the retina with areas of clustered cells and other areas with a shortage of cells. These movements lead to structural changes in the layers of the retinal that can be measured, with regions thickened versus other changes thinned over time as the disease progresses. All this changes can be detected by examining the retina in vivo with optical coherence tomography (OCT) in both animal experimental models and human. OCT allows the evaluation of cellular behaviour indirectly in neurodegeneration, in which at first a thickening can be observed that could be associated with protein deposits or cellular changes caused as a result of the inflammatory process, then in more advanced stages the tissue experiences a neurodegeneration, related to a thinning of the retina due to neuronal loss. These changes have been seen that are similar in both the experimental model, as in AD patients.Therefore, the microglia is not an innocent bystander, but a key player, generating changes based on its dual form of activation.

Enhanced Brain Retention of Aβ4‐x Proteoforms and its Contribution to Amyloid Deposits in Alzheimer’s Disease

The deposition of amyloid‐β (Aβ) in brain parenchyma and cerebral vasculature together with intraneuronal neurofibrillary tangles and the gradual loss of synapsis are central neuropathological hallmarks of Alzheimer’s disease (AD). Although it remains unclear what primarily triggers and drives the progression of AD, different lines of investigation point out to a central role of oligomeric Aβ conformations for the disease pathogenesis. Recent studies revealed that the molecular heterogeneity of Aβ deposits is significantly more complex than originally anticipated, extending well beyond the classic Aβ1‐40/Aβ1‐42 dichotomy and being substantially expanded by the presence of multiple post‐translational modifications that increase the proteome diversity. Although N‐terminal truncations at glutamates 3 and 11 and their subsequent cyclation to pyroglutamate are perhaps the most extensively studied modifications, many others have been reported, with species beginning at phenylalanine 4 and bearing an intact C‐terminus being especially relevant. Indeed, Aβ4‐42 peptides were reported as major components of amyloid plaque cores and the limited studies available seem to indicate their relative abundance in patients with AD, Down’s syndrome, and vascular dementia. In this work we assessed the topographic distribution of these species with in‐house‐generated anti‐Aβ4‐x monoclonal 18H6 – specific for Aβ species starting at position 4 and blind for the full‐length Aβ40 and Aβ42 – and used in conjunction with thioflavin S and antibodies specific for the C‐terminus 40 and 42 in confirmed AD cases. All types of Aβ deposits – parenchymal plaques, pre‐amyloid lesions and CAA – contained the Aβ epitopes tested, albeit expressed in different proportions. These co‐localizations were further stressed by combining double immunohistochemical analysis with Thioflavin S staining, showing the preferential location of Aβ4‐x species in CAA and cored plaques with fibrillar amyloid conformation, strongly suggesting poor clearance characteristics. To investigate the brain clearance of Aβ4‐x species in comparison with full‐length Aβ40 and Aβ42 homologues, peptides were synthesized, biochemically and biophysically characterized, radiolabeled with [125I]Na and utilized for in vivo brain clearance experiments in 5–6‐week‐old C57Bl/6 mice. Brain efflux of all monomeric Aβ species tested was fast, with &gt;80% cleared within the 60 min duration of the experiment whereas retention of oligomeric preparations was consistently higher than that of monomeric forms. Notably, clearance of Aβ4‐40 and Aβ4‐42 was significantly lower (&lt;45%) than the full‐length counterparts (61% ‐ 68%), further supporting a pathogenic role for Aβ oligomerization in AD and specifically highlighting the importance of oligomeric Aβ4‐x species exhibiting higher brain retention compared to their full‐length counterparts. Since the process of multimerization is concentration dependent, failing to remove these oligomeric forms of Aβ from the brain will further contribute to exacerbate the amyloidogenic loop and the persistence of amyloid deposits.

Identification of Multicolor Fluorescent Probes for Heterogeneous Aβ Deposits in Alzheimer's Disease.

Accumulation of amyloid-beta (Aβ) into amyloid plaques and hyperphosphorylated tau into neurofibrillary tangles (NFTs) are pathological hallmarks of Alzheimer’s disease (AD). There is a significant intra- and inter-individual variability in the morphology and conformation of Aβ aggregates, which may account in part for the extensive clinical and pathophysiological heterogeneity observed in AD. In this study, we sought to identify an array of fluorescent dyes to specifically probe Aβ aggregates, in an effort to address their diversity. We screened a small library of fluorescent probes and identified three benzothiazole-coumarin derivatives that stained both vascular and parenchymal Aβ deposits in AD brain sections. The set of these three dyes allowed the visualization of Aβ deposits in three different colors (blue, green and far-red). Importantly, two of these dyes specifically stained Aβ deposits with no apparent staining of hyperphosphorylated tau or α-synuclein deposits. Furthermore, this set of dyes demonstrated differential interactions with distinct types of Aβ deposits present in the same subject. Aβ aggregate-specific dyes identified in this study have the potential to be further developed into Aβ imaging probes for the diagnosis of AD. In addition, the far-red dye we identified in this study may serve as an imaging probe for small animal imaging of Aβ pathology. Finally, these dyes in combination may help us advance our understanding of the relation between the various Aβ deposits and the clinical diversity observed in AD.

In situ structural biology of pathological protein deposits in Alzheimer's disease

Fibrillar aggregates of Aβ peptides and tau protein are defining features of Alzheimer's disease (AD) and evidence is accumulating that peptide conformation within fibrils correlates with disease subtype. Nevertheless, high resolution structures of fibrillar polymorphs fall short of defining the molecular mechanisms underlying disease. Mapping of the spatial distribution of polymorphs at multiple length scales—within plaques and tangles, across brain tissue, and in multiple brain regions—is being carried out to supplement high resolution information.

&lt;i&gt;In vivo&lt;/i&gt; evaluation of novel near-infrared fluorescence probe THK565 for imaging amyloid β and tau protein deposits in Alzheimer‘s mouse models

&lt;i&gt;In vivo&lt;/i&gt; evaluation of novel near-infrared fluorescence probe THK565 for imaging amyloid β and tau protein deposits in Alzheimer‘s mouse models

Extracellular Amyloid Deposits in Alzheimer’s and Creutzfeldt–Jakob Disease: Similar Behavior of Different Proteins?

Neurodegenerative diseases are characterized by the deposition of specific protein aggregates, both intracellularly and/or extracellularly, depending on the type of disease. The extracellular occurrence of tridimensional structures formed by amyloidogenic proteins defines Alzheimer’s disease, in which plaques are composed of amyloid β-protein, while in prionoses, the same term “amyloid” refers to the amyloid prion protein. In this review, we focused on providing a detailed didactic description and differentiation of diffuse, neuritic, and burnt-out plaques found in Alzheimer’s disease and kuru-like, florid, multicentric, and neuritic plaques in human transmissible spongiform encephalopathies, followed by a systematic classification of the morphological similarities and differences between the extracellular amyloid deposits in these disorders. Both conditions are accompanied by the extracellular deposits that share certain signs, including neuritic degeneration, suggesting a particular role for amyloid protein toxicity.

Hyperspectral retinal imaging shows amyloid‐like deposits in preclinical Alzheimer’s disease

AbstractBackgroundBrain beta‐amyloid deposits in Alzheimer’s disease (AD) are associated with retinal beta‐amyloid deposits post‐mortem, yet, findings from imaging the live human retina provide inconclusive evidence due to confounding factors such as drusen found in age‐related macular degeneration [(AMD), (Kayabasi 2014, Koronyo‐Hamaoui 2017)]. We therefore aimed to identify drusen and retinal amyloid‐like deposits via hyperspectral imaging.MethodCognitively normal (n=45) older adults, aged 50‐85 years with known neocortical amyloid burden (NAB) including those at‐risk for AD (SUVR≥1.35), were recruited from our study cohorts in Sydney, New South Wales. All participants completed imaging in the spectral range of 450 to 900nm wavelength at 5nm steps. 11 participants completed optical coherence tomography to confirm presence/absence of drusen. Raw hyperspectral images were visually scanned to identify retinal deposits.ResultMatched to our reference images, we identified retinal hyperintense ‘nano dots’. The spectral range of drusen and nano dots was between 500 to 635 nm and 450 to 600 nm respectively. Drusen was more around the macula in a clustered fashion. nano dots were seen in abundance in the superior quadrant, scattered along the blood vessels and appeared smaller with well distinct margin. In participants with no history of AMD (n=30), the visual identification of nano dots was possible for 77% in high NAB and no nanodots were visually identified in 70% in low NAB group participants.ConclusionFurther investigation is required to examine the nano dots to determine whether they have diagnostic significance.

Proteasomal degradation of the intrinsically disordered protein tau at single-residue resolution.

Intrinsically disordered proteins (IDPs) can be degraded in a ubiquitin-independent process by the 20S proteasome. Decline in 20S activity characterizes neurodegenerative diseases. Here, we examine 20S degradation of IDP tau, a protein that aggregates into insoluble deposits in Alzheimer's disease. We show that cleavage of tau by the 20S proteasome is most efficient within the aggregation-prone repeat region of tau and generates both short, aggregation-deficient peptides and two long fragments containing residues 1 to 251 and 1 to 218. Phosphorylation of tau by the non-proline-directed Ca2+/calmodulin-dependent protein kinase II inhibits degradation by the 20S proteasome. Phosphorylation of tau by GSK3β, a major proline-directed tau kinase, modulates tau degradation kinetics in a residue-specific manner. The study provides detailed insights into the degradation products of tau generated by the 20S proteasome, the residue specificity of degradation, single-residue degradation kinetics, and their regulation by posttranslational modification.

N-terminal heterogeneity of parenchymal and vascular amyloid-β deposits in Alzheimer's disease.

Aims:The deposition of amyloid-β (Aβ) peptides in the form of extracellular plaques in the brain represents one of the classical hallmarks of Alzheimer’s disease (AD). In addition to ‘full-length’ Aβ starting with aspartic acid (Asp-1), considerable amounts of various shorter, N-terminally truncated Aβ peptides have been identified by mass spectrometry in autopsy samples from individuals with AD.Methods:Selectivity of several antibodies detecting full-length, total or N-terminally truncated Aβ species has been characterized with capillary isoelectric focusing assays using a set of synthetic Aβ peptides comprising different N-termini. We further assessed the N-terminal heterogeneity of extracellular and vascular Aβ peptide deposits in the human brain by performing immunohistochemical analyses using sporadic AD cases with antibodies targeting different N-terminal residues, including the biosimilar antibodies Bapineuzumab and Crenezumab.Results:While antibodies selectively recognizing Aβ1–x showed a much weaker staining of extracellular plaques and tended to accentuate cerebrovascular amyloid deposits, antibodies detecting Aβ starting with phenylalanine at position 4 of the Aβ sequence showed abundant amyloid plaque immunoreactivity in the brain parenchyma. The biosimilar antibody Bapineuzumab recognized Aβ starting at Asp-1 and demonstrated abundant immunoreactivity in AD brains.Discussion:In contrast to other studied Aβ1–x-specific antibodies, Bapineuzumab displayed stronger immunoreactivity on fixed tissue samples than with sodium dodecyl sulfate-denatured samples on Western blots. This suggests conformational preferences of this antibody. The diverse composition of plaques and vascular deposits stresses the importance of understanding the roles of various Aβ variants during disease development and progression in order to generate appropriate target-developed therapies.

Systemic LPS-induced A\u03b2-solubilization and clearance in A\u03b2PP-transgenic mice is diminished by heparanase overexpression

Amyloid-β (Aβ) is the main constituent of amyloid deposits in Alzheimer’s disease (AD). The neuropathology is associated with neuroinflammation. Here, we investigated effects of systemic lipopolysaccharide (LPS)-treatment on neuroinflammation and Aβ deposition in AβPP-mice and double-transgenic mice with brain expression of AβPP and heparanase, an enzyme that degrades HS and generates an attenuated LPS-response. At 13 months of age, the mice received a single intraperitoneal injection of 50 µg LPS or vehicle, and were sacrificed 1.5 months thereafter. Aβ in the brain was analyzed histologically and biochemically after sequential detergent extraction. Neuroinflammation was assessed by CD45 immunostaining and mesoscale cytokine/chemokine ELISA. In single-transgenic mice, LPS-treatment reduced total Aβ deposition and increased Tween-soluble Aβ. This was associated with a reduced CXCL1, IL-1β, TNF-α-level and microgliosis, which correlated with amyloid deposition and total Aβ. In contrast, LPS did not change Aβ accumulation or inflammation marker in the double-transgenic mice. Our findings suggest that a single pro-inflammatory LPS-stimulus, if given sufficient time to act, triggers Aβ-clearance in AβPP-transgenic mouse brain. The effects depend on HS and heparanase.

4',6-Diamidino-2-Phenylindole Distinctly Labels Tau Deposits.

Tau deposits have distinct biochemical characteristics and vary morphologically based on identification with tau antibodies and several chemical dyes. Here, we report 4',6-diamidino-2-phenylindole (DAPI)-positivity of tau deposits. Furthermore, we investigated the cause for this positivity. DAPI was positive in 3R/4R (3-repeat/4-repeat) tau deposits in Alzheimer's disease, myotonic dystrophy, and neurodegeneration with brain iron accumulation, and in 4R tau deposits in corticobasal degeneration, but negative in 4R tau deposits in frontotemporal dementia with parkinsonism-17 and progressive supranuclear palsy. The peak emission wavelength of DAPI after binding to a tau deposit was similar to that after binding to a nucleus. This DAPI-positivity was conspicuous at the optimum concentration of 2 μg/ml. DAPI-positivity was diminished after formic acid treatment, but preserved after nucleic acid elimination and phosphate moiety blocking. Our results suggest that staining with 2 μg/ml DAPI is a common but useful tool to differentially detect tau deposits in various tauopathies.

Measurement of Tau Filament Fragmentation Provides Insights into Prion-like Spreading.

The ordered assembly of amyloidogenic proteins causes a wide spectrum of common neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. These diseases share common features with prion diseases, in which misfolded proteins can self-replicate and transmit disease across different hosts. Deciphering the molecular mechanisms that underlie the amplification of aggregates is fundamental for understanding how pathological deposits can spread through the brain and drive disease. Here, we used single-molecule microscopy to study the assembly and replication of tau at the single aggregate level. We found that tau aggregates have an intrinsic ability to amplify by filament fragmentation, and determined the doubling times for this replication process by kinetic modeling. We then simulated the spreading time for aggregates through the brain and found this to be in good agreement with both the observed time frame for spreading of pathological tau deposits in Alzheimer's disease and in experimental models of tauopathies. With this work we begin to understand the physical parameters that govern the spreading rates of tau and other amyloids through the human brain.

Statistical evaluation of test-retest studies in PET brain imaging.

Positron emission tomography (PET) is a molecular imaging technology that enables in vivo quantification of metabolic activity or receptor density, among other applications. Examples of applications of PET imaging in neuroscience include studies of neuroreceptor/neurotransmitter levels in neuropsychiatric diseases (e.g., measuring receptor expression in schizophrenia) and of misfolded protein levels in neurodegenerative diseases (e.g., beta amyloid and tau deposits in Alzheimer’s disease). Assessment of a PET tracer’s test-retest properties is an important component of tracer validation, and it is usually carried out using data from a small number of subjects. Here, we investigate advantages and limitations of test-retest metrics that are commonly used for PET brain imaging, including percent test-retest difference and intraclass correlation coefficient (ICC). In addition, we show how random effects analysis of variance, which forms the basis for ICC, can be used to derive additional test-retest metrics, which are generally not reported in the PET brain imaging test-retest literature, such as within-subject coefficient of variation and repeatability coefficient. We reevaluate data from five published clinical PET imaging test-retest studies to illustrate the relative merits and utility of the various test-retest metrics. We provide recommendations on evaluation of test-retest in brain PET imaging and show how the random effects ANOVA based metrics can be used to supplement the commonly used metrics such as percent test-retest. Random effects ANOVA is a useful model for PET brain imaging test-retest studies. The metrics that ensue from this model are recommended to be reported along with the percent test-retest metric as they capture various sources of variability in the PET test-retest experiments in a succinct way.

Generation of Clickable Pittsburgh Compound B for the Detection and Capture of β-Amyloid in Alzheimer's Disease Brain.

The benzothiazole-aniline derivative Pittsburgh Compound B (PiB) is the prototypical amyloid affinity probe developed for the in vivo positron emission tomography (PET) detection of amyloid beta (Aβ) deposits in Alzheimer's disease (AD). Specific high-affinity binding sites for PiB have been found to vary among AD cases with comparable Aβ load, and they are virtually absent on human-sequence Aβ deposits in animal models, none of which develop the full phenotype of AD. PiB thus could be an informative probe for studying the pathobiology of Aβ, but little is known about the localization of PiB binding at the molecular or structural level. By functionalizing the 6-hydroxy position of PiB with a PEG3 spacer and a terminal alkyne (propargyl) moiety, we have developed a clickable PiB compound that was derivatized with commercially available azide-labeled fluorophores or affinity-tags using copper-catalyzed azide-alkyne cycloaddition reactions, commonly referred to as "click" chemistry. We have determined that both the clickable PiB derivative and its fluorescently labeled conjugate have low nanomolar binding affinities for synthetic Aβ aggregates. Furthermore, the fluorescent-PiB conjugate can effectively bind Aβ aggregates in human AD brain homogenates and tissue sections. By covalently coupling PiB to magnetic beads, Aβ aggregates were also affinity-captured from AD brain extracts. Thus, the clickable PiB derivative described herein can be used to generate a wide variety of covalent conjugates, with applications including the fluorescence detection of Aβ, the ultrastructural localization of PiB binding, and the affinity capture and structural characterization of Aβ and other cofactors from AD brains.