Alzheimer's Disease Brain Sections Research Articles

Automated deep learning segmentation of neuritic plaques and neurofibrillary tangles in Alzheimer disease brain sections using a proprietary software.

Neuropathological diagnosis of Alzheimer disease (AD) relies on semiquantitative analysis of phosphorylated tau-positive neurofibrillary tangles (NFTs) and neuritic plaques (NPs), without consideration of lesion heterogeneity in individual cases. We developed a deep learning workflow for automated annotation and segmentation of NPs and NFTs from AT8-immunostained whole slide images (WSIs) of AD brain sections. Fifteen WSIs of frontal cortex from 4 biobanks with varying tissue quality, staining intensity, and scanning formats were analyzed. We established an artificial intelligence (AI)-driven iterative procedure to improve the generation of expert-validated annotation datasets for NPs and NFTs thereby increasing annotation quality by >50%. This strategy yielded an expert-validated annotation database with 5013 NPs and 5143 NFTs. We next trained two U-Net convolutional neural networks for detection and segmentation of NPs or NFTs, achieving high accuracy and consistency (mean Dice similarity coefficient: NPs, 0.77; NFTs, 0.81). The workflow showed high generalization performance across different cases. This study serves as a proof-of-concept for the utilization of proprietary image analysis software (Visiopharm) in the automated deep learning segmentation of NPs and NFTs, demonstrating that AI can significantly improve the annotation quality of complex neuropathological features and enable the creation of highly precise models for identifying these markers in AD brain sections.

Aβ-Aggregation-Generated Blue Autofluorescence Illuminates Senile Plaques as well as Complex Blood and Vascular Pathologies in Alzheimer's Disease.

Senile plaque blue autofluorescence was discovered around 40 years ago, however, its impact on Alzheimer's disease (AD) pathology has not been fully examined. We analyzed senile plaques with immunohistochemistry and fluorescence imaging on AD brain sections and also Aβ aggregation in vitro. In DAPI or Hoechst staining, the nuclear blue fluorescence could only be correctly assigned after subtracting the blue plaque autofluorescence. The flower-like structures wrapping dense-core blue fluorescence formed by cathepsin D staining could not be considered central-nucleated neurons with defective lysosomes since there was no nuclear staining in the plaque core when the blue autofluorescence was subtracted. Both Aβ self-oligomers and Aβ/hemoglobin heterocomplexes generated blue autofluorescence. The Aβ amyloid blue autofluorescence not only labels senile plaques but also illustrates red cell aggregation, hemolysis, cerebral amyloid angiopathy, vascular plaques, vascular adhesions, and microaneurysms. In summary, we conclude that Aβ-aggregation-generated blue autofluorescence is an excellent multi-amyloidosis marker in Alzheimer's disease.

Comparison of Monoamine Oxidase-A, Aβ Plaques, Tau, and Translocator Protein Levels in Postmortem Human Alzheimer's Disease Brain.

Increased monoamine oxidase-A (MAO-A) activity in Alzheimer's disease (AD) may be detrimental to the point of neurodegeneration. To assess MAO-A activity in AD, we compared four biomarkers, Aβ plaques, tau, translocator protein (TSPO), and MAO-A in postmortem AD. Radiotracers were [18F]FAZIN3 for MAO-A, [18F]flotaza and [125I]IBETA for Aβ plaques, [124/125I]IPPI for tau, and [18F]FEPPA for TSPO imaging. Brain sections of the anterior cingulate (AC; gray matter GM) and corpus callosum (CC; white matter WM) from cognitively normal control (CN, n = 6) and AD (n = 6) subjects were imaged using autoradiography and immunostaining. Using competition with clorgyline and (R)-deprenyl, the binding of [18F]FAZIN3 was confirmed to be selective to MAO-A levels in the AD brain sections. Increases in MAO-A, Aβ plaque, tau, and TSPO activity were found in the AD brains compared to the control brains. The [18F]FAZIN3 ratio in AD GM versus CN GM was 2.80, suggesting a 180% increase in MAO-A activity. Using GM-to-WM ratios of AD versus CN, a >50% increase in MAO-A activity was observed (AD/CN = 1.58). Linear positive correlations of [18F]FAZIN3 with [18F]flotaza, [125I]IBETA, and [125I]IPPI were measured and suggested an increase in MAO-A activity with increases in Aβ plaques and tau activity. Our results support the finding that MAO-A activity is elevated in the anterior cingulate cortex in AD and thus may provide a new biomarker for AD in this brain region.

Fluorine-18-Labeled Diaryl-azines as Improved β-Amyloid Imaging Tracers: From Bench to First-in-Human Studies.

The deposition of β-amyloid (Aβ) in the brain is a pathologic hallmark of Alzheimer's disease (AD), appearing years before the onset of symptoms, and its detection is incorporated into clinical diagnosis. Here, we have discovered and developed a class of diaryl-azine derivatives for detecting Aβ plaques in the AD brain using PET imaging. After a set of comprehensive preclinical assessments, we screened out a promising Aβ-PET tracer, [18F]92, with a high binding affinity to the Aβ aggregates, significant binding ability with the AD brain sections, and optimal brain pharmacokinetic properties in rodents and non-human primates. The first-in-human PET study declared that [18F]92 displayed low white matter uptake and could bind to Aβ pathology for distinguishing AD from healthy control subjects. All these results support that [18F]92 might become a promising PET tracer for visualizing Aβ pathology in AD patients.

An ultra-sensitive immunoassay detects and quantifies soluble Aβ oligomers in human plasma.

Evidence strongly suggests that soluble oligomers of amyloid beta protein (oAβ) help initiate the pathogenic cascade of Alzheimer's disease (AD). To date, there have been no validated assays specific for detecting and quantifying oAβ in human blood. We developed an ultrasensitive oAβ immunoassay using a novel capture antibody (71A1) with N-terminal antibody 3D6 for detection that specifically quantifies soluble oAβ in the human brain, cerebrospinal fluid (CSF), and plasma. Two new antibodies (71A1; 1G5) are oAβ-selective, label Aβ plaques in non-fixed AD brain sections, and potently neutralize the synaptotoxicity of AD brain-derived oAβ. The 71A1/3D6 assay showed excellent dilution linearity in CSF and plasma without matrix effects, good spike recovery, and specific immunodepletion. We have created a sensitive, high throughput, and inexpensive method to quantify synaptotoxic oAβ in human plasma for analyzing large cohorts of aged and AD subjects to assess the dynamics of this key pathogenic species and response to therapy.

Spectral photokinetic conversion of the fluorescent probes BSB and K114 for improved detection of amyloid assemblies.

Cross-β-sheet-rich protein fibrils are infamous for their accumulation in the brains of patients diagnosed with a number of neurodegenerative diseases, including Alzheimer's disease (AD). Disease-relevant fibrils are a result of deviation of the proteins from their native structure to a misfolded state resulting in aggregation and formation of fibrils. In this study, we explored the phenomenon of light-induced fluorescence enhancement of amyloid assemblies stained with two amyloid probes (BSB and K114) using Bombyx mori silk and human AD brain sections. The photoconversion effect, accompanied by an increase in fluorescence intensity and spectral blue-shift, was highly dependent on the chemical structures of the dyes, pH, presence of glycerol and the type of amyloid. The degree of intensity and spectral change over time in response to high laser exposure were quantified and analyzed using custom-written analysis tools. Our findings provide further insight into possible mechanisms of amyloid-mediated photoconversion kinetics of K114 and BSB, and may provide more insight into the molecular nature of various amyloid assemblies.

A New Highly Deuterated [18F]AV-45, [18F]D15FSP, for Imaging β-Amyloid Plaques in the Brain.

[18F]AV-45 (florbetapir f18, Amyvid) is an FDA-approved PET imaging agent targeting Aβ plaques in the brain for diagnosis of Alzheimer's disease (AD). Its metabolites led to a high background in the brain and large bone uptake of [18F]F-, produced from dealkylation of the PEG chain. To slow down the in vivo metabolism, we report the design, synthesis, and evaluation of a highly deuterated derivative, [18F]D15FSP, and compared it with N-methyl-deuterated [18F]D3FSP and nondeuterated [18F]AV-45. D15FSP displayed excellent binding affinity (K i = 7.52 nM) to Aβ aggregates. In vitro autoradiography of [18F]D15FSP, [18F]D3FSP, and [18F]AV-45 showed excellent binding to Aβ plaques in human AD brain sections. Biodistribution studies displayed lower bone uptake at 120 min for [18F]D15FSP compared to that for [18F]D3FSP and [18F]AV-45 (1.44 vs 4.23 and 4.03%ID/g, respectively). As the highly deuterated [18F]D15FSP displayed excellent Aβ binding affinity, high initial brain penetration, and lower bone retention, it might be suitable for PET imaging in detecting Aβ plaques.

"Turn-On" Quinoline-Based Fluorescent Probe for Selective Imaging of Tau Aggregates in Alzheimer's Disease: Rational Design, Synthesis, and Molecular Docking.

Tau aggregation is believed to have a strong association with the level of cognitive deficits in Alzheimer's disease (AD). Thus, optical brain imaging of tau aggregates has recently gained substantial attention as a promising tool for the early diagnosis of AD. However, selective imaging of tau aggregates is a major challenge due to sharing similar β-sheet structures with homologous Aβ fibrils. Herein, four quinoline-based fluorescent probes (Q-tau) were judiciously designed using the donor-acceptor architecture for selective imaging of tau aggregates. In particular, probe Q-tau 4 exhibited a strong intramolecular charge transfer and favorable photophysical profile, such as a large Stokes' shift and fluorescence emission wavelength of 630 nm in the presence of tau aggregates. The probe also displayed a "turn-on" fluorescence behavior toward tau fibrils with a 3.5-fold selectivity versus Aβ fibrils. In addition, Q-tau 4 exhibited nanomolar binding affinity to tau aggregates (Kd = 16.6 nM), which was 1.4 times higher than that for Aβ fibrils. The mechanism of "turn-on" fluorescence was proposed to be an environment-sensitive molecular rotor-like response. Moreover, ex vivo labeling of human AD brain sections demonstrated favorable colocalization of Q-tau 4 and the phosphorylated tau antibody, while comparable limited staining was observed with Aβ fibrils. Molecular docking was conducted to obtain insights into the tau-binding mode of the probe. Collectively, Q-tau 4 has successfully been used as a tau-specific fluorescent imaging agent with lower background interference.

Characterization and Optimization of Benzimidazopyrimidine and Pyridoimidazopyridine Derivatives as Tau-SPECT Probes.

The accumulation of hyperphosphorylated tau protein in the brain is regarded as one of the hallmarks of Alzheimer's disease (AD). In vivo imaging of tau aggregates is helpful for diagnosis and monitoring of the progression of AD. In this study, we designed and synthesized novel radioiodinated benzimidazopyrimidine (BIPM) and pyridoimidazopyridine (PIP) derivatives with a monomethylamino, monoethylamino, monopropylamino, or diethylamino group as tau imaging probes for single-photon-emission computed tomography (SPECT). On in vitro autoradiography with AD brain sections, [125I]PIP-NHMe showed the highest selective binding affinity for tau aggregates among the radioiodinated BIPM and PIP derivatives. In a biodistribution study using normal mice, [125I]PIP-NHMe and [125I]PIP-NHEt displayed high initial uptake (6.62 and 6.86% ID/g, respectively, at 2 min postinjection) into and rapid clearance from the brain, with brain2min/brain30min ratios of 38.9 and 28.6, respectively. These results suggest that [123I]PIP-NHMe may be a novel SPECT probe that is useful for detecting tau aggregates in the AD brain.

Structure-Activity and Brain Kinetics Relationships of 18F-Labeled Benzimidazopyridine Derivatives as Tau PET Tracers.

Noninvasive imaging of tau aggregates with a positron emission tomography (PET) tracer is useful for the diagnosis and staging of Alzheimer's disease (AD). Recently, we found that benzimidazopyridine (BIP) is an attractive scaffold for developing PET and single photon computed emission tomography tracers targeting tau aggregates. In this study, we designed and synthesized five novel 18F-labeled compounds with various substituted groups or atoms at the 7-position of the BIP scaffold. In in vitro autoradiographic studies, all 18F-labeled BIP derivatives selectively bound to tau aggregates deposited in AD brain sections. On the other hand, the initial brain uptake of these compounds was affected by the type of substituted group or halogen atom introduced into the 7-position of the BIP scaffold. Among these compounds, [18F]Me-BIPF showed the highest brain uptake (6.79% ID/g at 2 min postinjection) and 2 min/60 min ratio (3.59). These results suggest that appropriate introduction of the substituted group or atom into the 7-position of the BIP scaffold may be effective for developing useful tau PET tracers.

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

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

Synthesis and evaluation of 2-pyrrolopyridinylquinoline derivatives as selective tau PET tracers for the diagnosis of Alzheimer's disease

Introduction[18F]THK-5351 was originally developed as a positron emission tomography (PET) imaging tracer for the detection of accumulated tau proteins, the pathological hallmark of Alzheimer's disease (AD). However, clinical studies of [18F]THK-5351 revealed the existence of off-target binding to monoamine oxidase-B (MAO-B). To overcome this off-target binding, in this work, we synthesized and evaluated 2-pyrrolopyridinylquinoline (PPQ) derivatives as selective tau PET imaging tracers. MethodsThe core structure of PPQ derivatives was synthesized mainly using the Buchwald-Hartwig amination coupling reaction. All derivatives were evaluated for binding affinity towards tau and MAO-B by in vitro competitive binding assay. Radiosynthesis of PPQ derivatives was performed by 18F-radiolabeling of their tosylate precursors with activated [18F]KF/Kryptofix222 complex in dimethylsulfoxide by heating at 110 °C for 10 min. The biological properties of these [18F]PPQ derivatives were characterized by in vitro autoradiography of postmortem AD brain sections and by assay of ex vivo biodistribution in mice. ResultsThe PPQ derivatives were synthesized, with yields of 49–84%. In vitro competitive binding assay revealed that two novel PPQ derivatives—PPQ8 and PPQ9—demonstrated high binding affinity for tau (IC50 = 4.9 and 6.9 nM, respectively). The radiosynthesis of [18F]PPQ8 and [18F]PPQ9 yielded 1.4% and 50.1% isolated non-decay corrected radiochemical yield, respectively, with >99% radiochemical purity. The molar radioactivities of [18F]PPQ8 and [18F]PPQ9 were 16.9 and 64.8 GBq/μmol, respectively. The in vitro and ex vivo biological characterization of [18F]PPQ8 and [18F]PPQ9 revealed that these tracers were selective for tau in AD brain sections without off-target binding, and they furthermore demonstrated brain uptake in normal mice. Conclusions18F-labeled PPQ derivatives improved binding affinity and selectivity for tau aggregates in AD. Further structural optimization to improve pharmacokinetics for potent tau PET imaging tracers is required.

18F-SMBT-1: A Selective and Reversible PET Tracer for Monoamine Oxidase-B Imaging.

Reactive astrocytes play a key role in the pathogenesis of various neurodegenerative diseases. Monoamine oxidase-B (MAO-B) is one of the promising targets for the imaging of astrogliosis in the human brain. A novel selective and reversible MAO-B tracer, (S)-(2-methylpyrid-5-yl)-6-[(3-18F-fluoro-2-hydroxy)propoxy]quinoline (18F-SMBT-1), was successfully developed via lead optimization from the first-generation tau PET tracer 18F-THK-5351. Methods: SMBT-1 was radiolabeled with 18F using the corresponding precursor. The binding affinity of radiolabeled compounds to MAO-B was assessed using saturation and competitive binding assays. The binding selectivity of 18F-SMBT-1 to MAO-B was evaluated by autoradiography of frozen human brain tissues. The pharmacokinetics and metabolism were assessed in normal mice after intravenous administration of 18F-SMBT-1. A 14-d toxicity study after the intravenous administration of 18F-SMBT-1 was performed using rats and mice. Results: In vitro binding assays demonstrated a high binding affinity of 18F-SMBT-1 to MAO-B (dissociation constant, 3.7 nM). In contrast, it showed low binding affinity to MAO-A and protein aggregates such as amyloid-β and tau fibrils. Autoradiographic analysis showed higher amounts of 18F-SMBT-1 binding in the Alzheimer disease brain sections than in the control brain sections. 18F-SMBT-1 binding was completely displaced with the reversible MAO-B inhibitor lazabemide, demonstrating the high selectivity of 18F-SMBT-1 for MAO-B. Furthermore, 18F-SMBT-1 showed a high uptake by brain, rapid washout, and no radiolabeled metabolites in the brain of normal mice. 18F-SMBT-1 showed no significant binding to various receptors, ion channels, or transporters, and no toxic effects related to its administration were observed in mice and rats. Conclusion:18F-SMBT-1 is a promising and selective MAO-B PET tracer candidate, which would be useful for quantitative monitoring of astrogliosis in the human brain.

SELECTIVE TARGETING OF AMYLOID-BETA OLIGOMER SPECIES BY PMN310, A MONOCLONAL ANTIBODY RATIONALLY DESIGNED FOR GREATER THERAPEUTIC POTENCY IN ALZHEIMER’S DISEASE

Advances in the understanding of Alzheimer's disease (AD) suggest that pathogenesis is not directly related to plaque burden, but rather to soluble toxic amyloid-beta oligomers (AßO). Therapeutic antibodies targeting Aß monomers and/or plaque have shown limited efficacy and dose-limiting adverse events in clinical trials. These findings suggest that antibodies capable of selectively neutralizing toxic AßO may achieve improved efficacy and safety. To this end, we generated a monoclonal antibody, PMN310, against a conformational Aß epitope predicted by computational modeling to be exposed on toxic AßO but not monomers or fibrils. The binding profile of PMN310 was characterized by surface plasmon resonance (SPR) and immunohistochemistry (IHC). Its ability to neutralize the propagation and toxicity of AßO was assessed in vitro in a thioflavin-T propagation assay and in cultures of primary rodent neurons, respectively. Protection against memory loss was tested in wild-type mice injected intracerebroventricularly with AßO. Brain exposure and kinetics were evaluated in aged wild-type mice and APP/PS1 transgenic mice. SPR analysis showed preferential binding of PMN310 to synthetic Ab oligomers vs monomers. Testing of soluble human AD brain extracts fractionated by size-exclusion chromatography consistently showed greater binding of PMN310 to the toxic oligomer-enriched low molecular weight fraction (<70 kDa) compared to aducanumab and bapineuzumab. IHC on frozen human AD brain sections showed no immunoreactivity of PMN310 with Aß deposits. In contrast, clear binding of parenchymal and vascular Aß was observed with aducanumab and bapineuzumab, consistent with the clinical occurrence of ARIA associated with these antibodies. In in vitro activity assays, PMN310 inhibited AßO propagation and neuronal toxicity. In vivo, PMN310 prevented AßO-induced loss of memory formation and reduced synaptic loss and inflammation. Systemic administration of PMN310 in mice resulted in brain exposure and kinetics comparable to those of other therapeutic human monoclonal antibodies. The greater selectivity of PMN310 for AßO and the potential to safely administer high doses with a reduced risk of ARIA suggest that it is likely to achieve greater therapeutic potency compared to other Aß-directed antibodies. Humanized PMN310 is currently in advanced preclinical development.

Improved automated synthesis of [18F]FINH-Me via direct radio-fluorination and quality control for Aβ-amyloid imaging.

Alzheimer's disease (AD) is a neurological disorder that leads to a loss of cognitive functioning, affecting older people as well as their families. An early detection of AD is therefore crucial to help people maintain mental function, and slow down or delay the symptoms of the disease. Studies have confirmed that the deposition of the amyloid-β peptide in the brain is a central event in AD pathology. An imaging probe for Aβmight provide a useful tool to be used in the diagnosis and monitoring response to therapy of AD. This article reports a new AD imaging probe [18F]FINH-Me, which has a high molar activity for Aβ. Automated synthesis of [18F]FINH-Me was performed in a single reactor using a two-step procedure; fluorination and hydrolysis. The qualities of this probe were tested, and autoradiography was performed to determine if the probe was bound to the AD sections. And the micro-PET/CT imaging experiment was done to further confirm the combination of the probe and Aβ. Optimized synthesis of [18F]FINH-Me was achieved in 90 minutes. The probe was then scaled up for large-batch production to generate 18.79-23.53 GBq of [18F]FINH-Me at EOS (N.=15) in high molar activity (79.9-122 GBq/µmol, at EOS, N.=15). In the AD brain sections, the radio activity was concentrated at the hippocampus and the temporal lobe area. [18F]FINH-Me is a promising PET probe to be used in AD diagnose.

Curcumin restores innate immune Alzheimer's disease risk gene expression to ameliorate Alzheimer pathogenesis.

Alzheimer's disease (AD) genetics implies a causal role for innate immune genes, TREM2 and CD33, products that oppose each other in the downstream Syk tyrosine kinase pathway, activating microglial phagocytosis of amyloid (Aβ). We report effects of low (Curc-lo) and high (Curc-hi) doses of curcumin on neuroinflammation in APPsw transgenic mice. Results showed that Curc-lo decreased CD33 and increased TREM2 expression (predicted to decrease AD risk) and also increased TyroBP, which controls a neuroinflammatory gene network implicated in AD as well as phagocytosis markers CD68 and Arg1. Curc-lo coordinately restored tightly correlated relationships between these genes' expression levels, and decreased expression of genes characteristic of toxic pro-inflammatory M1 microglia (CD11b, iNOS, COX-2, IL1β). In contrast, very high dose curcumin did not show these effects, failed to clear amyloid plaques, and dysregulated gene expression relationships. Curc-lo stimulated microglial migration to and phagocytosis of amyloid plaques both in vivo and in ex vivo assays of sections of human AD brain and of mouse brain. Curcumin also reduced levels of miR-155, a micro-RNA reported to drive a neurodegenerative microglial phenotype. In conditions without amyloid (human microglial cells in vitro, aged wild-type mice), Curc-lo similarly decreased CD33 and increased TREM2. Like curcumin, anti-Aβ antibody (also reported to engage the Syk pathway, increase CD68, and decrease amyloid burden in human and mouse brain) increased TREM2 in APPsw mice and decreased amyloid in human AD sections ex vivo. We conclude that curcumin is an immunomodulatory treatment capable of emulating anti-Aβ vaccine in stimulating phagocytic clearance of amyloid by reducing CD33 and increasing TREM2 and TyroBP, while restoring neuroinflammatory networks implicated in neurodegenerative diseases.

Structure-Activity Relationships of Radioiodinated Benzoimidazopyridine Derivatives for Detection of Tau Pathology.

It is generally accepted that neurofibrillary tangles consisting of tau proteins are involved in the pathogenesis of Alzheimer's disease (AD). For selective detection of tau pathology, we synthesized and evaluated radioiodinated benzoimidazopyridine (BIP) derivatives with an alkylamino group as tau imaging probes. In vitro selectivity to tau aggregates and in vivo pharmacokinetics of BIP derivatives varied markedly, being strongly dependent on the alkylamino group. In in vitro autoradiography with AD brain sections, the BIP derivative with a dimethylamino group (BIP-NMe2) showed the highest selectivity to tau aggregates. Regarding the biodistribution using normal mice, the BIP derivative with an ethylamino group (BIP-NHEt) showed the highest uptake (6.04% ID/g at 2 min postinjection) into and rapid washout (0.12% ID/g at 60 min postinjection) from the brain. These results suggest that the introduction of an optimal alkylamino group into the BIP scaffold may lead to the development of more potential tau imaging probes.

Development of SPECT Probes for In Vivo Imaging of β-Amyloid and Tau Aggregates in the Alzheimer's Disease Brain

Alzheimer's disease (AD) is the most common form of irreversible dementia among elderly people. In the postmortem brains of AD patients, the deposition of senile plaques composed of β-amyloid (Aβ) peptides and neurofibrillary tangles composed of highly phosphorylated tau proteins are two neuropathological hallmarks. Therefore, the in vivo imaging of Aβ and tau aggregates with positron-emission tomography (PET) or single-photon emission computed tomography (SPECT) would promote drug development, early diagnosis, and monitoring of the disease status in AD patients. In this study, we designed and synthesized novel Aβ and tau imaging probes for SPECT. [125I]PBOX-3, developed as an Aβ imaging probe, showed high affinity for Aβ aggregates in vitro. A SPECT/CT study with [123I]PBOX-3 revealed a higher level of radioactivity in a Tg2576 mouse, which is the AD model mouse, than in a wild-type mouse. In addition, ex vivo autoradiograms of brain sections from a Tg2576 mouse after the injection of [123I]PBOX-3 showed the selective binding of Aβ plaques. BIP-NMe2, developed as a tau imaging probe, showed high and selective affinity for tau aggregates in AD brain sections. In addition, [125I]BIP-NMe2 displayed high initial uptake into, and fast washout from, the normal mouse brain, suggesting that [125I]BIP-NMe2 has favorable pharmacokinetics for the in vivo imaging of tau aggregates. Taken together, we successfully developed an Aβ imaging probe, PBOX-3, and a tau imaging probe, BIP-NMe2. These probes may be used to develop novel methods for the diagnosis, treatment and monitoring of AD progression.

Synthesis and biological evaluation of curcumin analogs as β-amyloid imaging agents.

Detection of β-amyloid (Aβ) plaques in the brain is a very promising biomarker approach for early diagnosis of Alzheimer's disease (AD). A series of curcumin analogs (1,5-diphenyl-1,4-pentadien-3-one derivatives) were synthesized and evaluated. Specific binding to Aβ plaques was demonstrated in vitro using postmortem AD homogenates, and the fluorescent staining and autoradiography in vitro of postmortem AD brain sections were performed. Some compounds showed high binding affinities with Aβ plaques. Fluorescent staining indicated that compound 4e clearly stained Aβ plaques within AD brain sections. In biodistribution, radioiodinated ligand [125I]4e exhibited high brain uptake and favorable clearance from the brain. Autoradiography in vitro further confirmed the high affinities of [125I]4e. The results strongly suggested that [125I]4e might be developed into potential amyloid imaging agent for the detection of senile plaques in AD. [Formula: see text].

A COMPETITIVE EX-VIVO SCREENING ASSAY TO DETERMINE ABETA ANTIBODY MEDIATED PHAGOCYTOSIS AND PLAQUE SPECIFICITY

Extracellular amyloid plaque is one of the pathological hallmarks of Alzheimer's disease (AD). Up to 75% of the insoluble Aβ in AD brain is N-terminally truncated or modified, including Aβp3–42, which has only been detected in plaque. We reported previously the development of a monoclonal antibody that specifically targets Aβp3–42. The antibody showed robust clearance of pre-existing plaque without causing microhemorrhage (DeMattos et al, Neuron 2012). The Aβp3–42 antibodies with either minimal (IgG1 isotype) or maximal (IgG2a) effector function strongly labeled deposited plaque in both AD and PDAPP brain sections and led to significant reduction of Aβ1–42 in ex vivo phagocytosis assay. For screening and identification of future plaque-specific antibodies, we developed a functional antibody mediated phagocytosis assay to identify antibodies that would not cross react with full length Aβ and potentially be saturated by the soluble pool. We examined plaque specificity of Aβ antibodies by pre-incubating soluble Aβ1–40 with the antibodies prior to ex vivo phagocytosis assay with AD brain sections and murine microglia. Amyloid plaques were then fully solubilized and reduced to monomers by formic acid and Aβ peptides were resolved by acid urea gel electrophoresis coupled with western blotting analysis. Changes in distinctive Aβ species were then quantified. Using this method, we examined the plaque specific humanized Aβp3–42 antibody as well as the comparator 3D6 antibody which recognizes plaque as well as soluble Aβ. Consistent with our previous finding with murine Aβp3–42 antibody, humanized Aβp3–42 antibody significantly enhanced Aβ clearance via microglia-mediated phagocytosis. This effect was not influenced by pre-incubation of the antibody with soluble Aβ1–40 peptide, as revealed by acid urea gel electrophoresis, reflecting the plaque-specific characteristics of Aβp3–42 antibody. Humanized 3D6 antibody also invoked significant Aβ clearance. However, this effect was largely blocked by soluble Aβ1–40, reflecting the non-plaque specific nature of 3D6. We have developed an ex vivo functional assay for evaluating plaque specificity of Aβ antibodies. The assay will facilitate screening of future generation antibodies that target amyloid plaque.