Seeding competent TDP-43 persists in human patient and mouse muscle.

TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein that accumulates as aggregates in the central nervous system of some neurodegenerative diseases. However, TDP-43 aggregation is also a sensitive and specific pathologic feature found in a family of degenerative muscle diseases termed inclusion body myopathy (IBM). TDP-43 aggregates from ALS and FTD brain lysates may serve as self-templating aggregate seeds in vitro and in vivo, supporting a prion-like spread from cell to cell. Whether a similar process occurs in IBM patient muscle is not clear. We developed a mouse model of inducible, muscle-specific cytoplasmic localized TDP-43. These mice develop muscle weakness with robust accumulation of insoluble and phosphorylated sarcoplasmic TDP-43, leading to eosinophilic inclusions, altered proteostasis and changes in TDP-43-related RNA processing that resolve with the removal of doxycycline. Skeletal muscle lysates from these mice also have seeding competent TDP-43, as determined by a FRET-based biosensor, that persists for weeks upon resolution of TDP-43 aggregate pathology. Human muscle biopsies with TDP-43 pathology also contain TDP-43 aggregate seeds. Using lysates from muscle biopsies of patients with IBM, IMNM and ALS we found that TDP-43 seeding capacity was specific to IBM. Surprisingly, TDP-43 seeding capacity anti-correlated with TDP-43 aggregate and vacuole abundance. These data support that TDP-43 aggregate seeds are present in IBM skeletal muscle and represent a unique TDP-43 pathogenic species not previously appreciated in human muscle disease.

VCP Inhibition Augments NLRP3 Inflammasome Activation.

Lysosomal membrane permeabilization caused either via phagocytosis of particulates or the uptake of protein aggregates can trigger the activation of NLRP3 inflammasome- an intense inflammatory response that drives the release of the pro-inflammatory cytokine IL-1ß by regulating the activity of CASPASE 1. The maintenance of lysosomal homeostasis and lysosomal membrane integrity is facilitated by the AAA+ ATPase, VCP/p97 (VCP). However, the relationship between VCP and NLRP3 inflammasome activity remains unexplored. Here, we demonstrate that the VCP inhibitors, DBeQ and ML240 elicit the activation of NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs) when used as activation stimuli. Moreover, genetic inhibition of VCP or VCP chemical inhibition enhances lysosomal membrane damage and augments LLoME-associated NLRP3 inflammasome activation in BMDMs. Similarly, VCP inactivation also augments NLRP3 inflammasome activation mediated by aggregated alpha-synuclein fibrils and lysosomal damage. These data suggest that VCP is a participant in the complex regulation of NLRP3 inflammasome activation.

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. Here we develop and validate a method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and use solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise a mixture of single protofilament and two protofilament fibrils with very low twist. The protofilament fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural characterization of LBD Asyn fibrils and approaches for studying disease mechanisms, imaging agents and therapeutics targeting Asyn.

13C and 15N resonance assignments of alpha synuclein fibrils amplified from Lewy Body Dementia tissue.

Fibrils of the protein α-synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of 13C, 15N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.

13C and 15N Resonance Assignments of Alpha Synuclein Fibrils Amplified from Lewy Body Dementia Tissue.

Fibrils of the protein α-synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of 13C, 15N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise two protofilaments with pseudo-21 helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.

Aggregated alpha-synuclein transcriptionally activates pro-inflammatory canonical and non-canonical NF-κB signaling pathways in peripheral monocytic cells.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by chronic neuroinflammation, loss of dopaminergic neurons in the substantia nigra, and in several cases accumulation of alpha-synuclein fibril (α-syn) containing Lewy-bodies (LBs). Peripheral inflammation may play a causal role in inducing and perpetuating neuroinflammation in PD and accumulation of fibrillar α-syn has been reported at several peripheral sites including the gut and liver. Peripheral fibrillar α-syn may induce activation of monocytes via recognition by toll-like receptors (TLRs) and stimulation of downstream NF-κB signaling; however, the specific mechanism by which this occurs is not defined. In this study we utilized the THP-1 monocytic cell line to model the peripheral transcriptional response to preformed fibrillar (PFF) α-syn. Compared to monomeric α-syn, PFF α-syn displays overt inflammatory gene upregulation and pathway activation including broad pan-TLR signaling pathway activation and increases in TNF and IL1B gene expression. Notably, the non-canonical NF-κB signaling pathway gene and PD genome wide association study (GWAS) candidate NFKB2 was upregulated. Additionally, non-canonical NF-κB activation-associated RANK and CD40 pathways were also upregulated. Transcriptional-phenotype analysis suggests PFFs induce transcriptional programs associated with differentiation of monocytes towards macrophages and osteoclasts via non-canonical NF-κB signaling as a potential mechanism in which myeloid/monocyte cells may contribute to peripheral inflammation and pathogenesis in PD.

VCP suppresses proteopathic seeding in neurons.

BACKGROUND: Neuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery participating in this process is poorly understood. One proteinopathy called multisystem proteinopathy (MSP) is associated with dominant mutations in Valosin Containing Protein (VCP). MSP patients have muscle and neuronal degeneration characterized by aggregate pathology that can include αS, Tau and TDP-43. METHODS: We performed a fluorescent cell sorting based genome-wide CRISPR-Cas9 screen in αS biosensors. αS and TDP-43 seeding activity under varied conditions was assessed using FRET/Flow biosensor cells or immunofluorescence for phosphorylated αS or TDP-43 in primary cultured neurons. We analyzed in vivo seeding activity by immunostaining for phosphorylated αS following intrastriatal injection of αS seeds in control or VCP disease mutation carrying mice. RESULTS: One hundred fifty-four genes were identified as suppressors of αS seeding. One suppressor, VCP when chemically or genetically inhibited increased αS seeding in cells and neurons. This was not due to an increase in αS uptake or αS protein levels. MSP-VCP mutation expression increased αS seeding in cells and neurons. Intrastriatal injection of αS preformed fibrils (PFF) into VCP-MSP mutation carrying mice increased phospho αS expression as compared to control mice. Cells stably expressing fluorescently tagged TDP-43 C-terminal fragment FRET pairs (TDP-43 biosensors) generate FRET when seeded with TDP-43 PFF but not monomeric TDP-43. VCP inhibition or MSP-VCP mutant expression increases TDP-43 seeding in TDP-43 biosensors. Similarly, treatment of neurons with TDP-43 PFFs generates high molecular weight insoluble phosphorylated TDP-43 after 5 days. This TDP-43 seed dependent increase in phosphorlyated TDP-43 is further augmented in MSP-VCP mutant expressing neurons. CONCLUSION: Using an unbiased screen, we identified the multifunctional AAA ATPase VCP as a suppressor of αS and TDP-43 aggregate seeding in cells and neurons. VCP facilitates the clearance of damaged lysosomes via lysophagy. We propose that VCP's surveillance of permeabilized endosomes may protect against the proteopathic spread of pathogenic protein aggregates. The spread of distinct aggregate species may dictate the pleiotropic phenotypes and pathologies in VCP associated MSP.

Quantifying regional α -synuclein, amyloid ß, and tau accumulation in lewy body dementia.

OBJECTIVE: Parkinson disease (PD) is defined by the accumulation of misfolded α-synuclein (α-syn) in Lewy bodies and Lewy neurites. It affects multiple cortical and subcortical neuronal populations. The majority of people with PD develop dementia, which is associated with Lewy bodies in neocortex and referred to as Lewy body dementia (LBD). Other neuropathologic changes, including amyloid ß (Aß) and tau accumulation, occur in some LBD cases. We sought to quantify α-syn, Aß, and tau accumulation in neocortical, limbic, and basal ganglia regions. METHODS: We isolated insoluble protein from fresh frozen postmortem brain tissue samples for eight brains regions from 15 LBD, seven Alzheimer disease (AD), and six control cases. We measured insoluble α-syn, Aß, and tau with recently developed sandwich ELISAs. RESULTS: We detected a wide range of insoluble α-syn accumulation in LBD cases. The majority had substantial α-syn accumulation in most regions, and dementia severity correlated with neocortical α-syn. However, three cases had low neocortical levels that were indistinguishable from controls. Eight LBD cases had substantial Aß accumulation, although the mean Aß level in LBD was lower than in AD. The presence of Aß was associated with greater α-syn accumulation. Tau accumulation accompanied Aß in only one LBD case. INTERPRETATION: LBD is associated with insoluble α-syn accumulation in neocortical regions, but the relatively low neocortical levels in some cases suggest that other changes contribute to impaired function, such as loss of neocortical innervation from subcortical regions. The correlation between Aß and α-syn accumulation suggests a pathophysiologic relationship between these two processes.

APOE genotype regulates pathology and disease progression in synucleinopathy.

Apolipoprotein E (APOE) ε4 genotype is associated with increased risk of dementia in Parkinson's disease (PD), but the mechanism is not clear, because patients often have a mixture of α-synuclein (αSyn), amyloid-ß (Aß), and tau pathologies. APOE ε4 exacerbates brain Aß pathology, as well as tau pathology, but it is not clear whether APOE genotype independently regulates αSyn pathology. In this study, we generated A53T αSyn transgenic mice (A53T) on Apoe knockout (A53T/EKO) or human APOE knockin backgrounds (A53T/E2, E3, and E4). At 12 months of age, A53T/E4 mice accumulated higher amounts of brainstem detergent-insoluble phosphorylated αSyn compared to A53T/EKO and A53T/E3; detergent-insoluble αSyn in A53T/E2 mice was undetectable. By immunohistochemistry, A53T/E4 mice displayed a higher burden of phosphorylated αSyn and reactive gliosis compared to A53T/E2 mice. A53T/E2 mice exhibited increased survival and improved motor performance compared to other APOE genotypes. In a complementary model of αSyn spreading, striatal injection of αSyn preformed fibrils induced greater accumulation of αSyn pathology in the substantia nigra of A53T/E4 mice compared to A53T/E2 and A53T/EKO mice. In two separate cohorts of human patients with PD, APOE ε4/ε4 individuals showed the fastest rate of cognitive decline over time. Our results demonstrate that APOE genotype directly regulates αSyn pathology independent of its established effects on Aß and tau, corroborate the finding that APOE ε4 exacerbates pathology, and suggest that APOE ε2 may protect against αSyn aggregation and neurodegeneration in synucleinopathies.

Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation.

Aggregates of the RNA-binding protein TDP-43 (TAR DNA-binding protein) are a hallmark of the overlapping neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The process of TDP-43 aggregation remains poorly understood, and whether it includes formation of intermediate complexes is unknown. Here, we analyzed aggregates derived from purified TDP-43 under semidenaturing conditions, identifying distinct oligomeric complexes at the initial time points before the formation of large aggregates. We found that this early oligomerization stage is primarily driven by TDP-43's RNA-binding region. Specific binding to GU-rich RNA strongly inhibited both TDP-43 oligomerization and aggregation, suggesting that RNA interactions are critical for maintaining TDP-43 solubility. Moreover, we analyzed TDP-43 liquid-liquid phase separation and detected similar detergent-resistant oligomers upon maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, the ALS-linked TDP-43 mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that aggregates generated from purified TDP-43 seed intracellular aggregation detected by established TDP-43 pathology markers. Remarkably, cytoplasmic aggregate seeding was detected earlier for the A315T and M337V variants and was 50% more widespread than for WT TDP-43 aggregates. We provide evidence for an initial step of TDP-43 self-assembly into intermediate oligomeric complexes, whereby these complexes may provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.

Parkinson's disease and multiple system atrophy have distinct α-synuclein seed characteristics.

Parkinson's disease (PD) and multiple system atrophy (MSA) are distinct clinical syndromes characterized by the pathological accumulation of α-synuclein (α-syn) protein fibrils in neurons and glial cells. These disorders and other neurodegenerative diseases may progress via prion-like mechanisms. The prion model of propagation predicts the existence of "strains" that link pathological aggregate structure and neuropathology. Prion strains are aggregated conformers that stably propagate in vivo and cause disease with defined incubation times and patterns of neuropathology. Indeed, tau prions have been well defined, and research suggests that both α-syn and ß-amyloid may also form strains. However, there is a lack of studies characterizing PD- versus MSA-derived α-syn strains or demonstrating stable propagation of these unique conformers between cells or animals. To fill this gap, we used an assay based on FRET that exploits a HEK293T "biosensor" cell line stably expressing α-syn (A53T)-CFP/YFP fusion proteins to detect α-syn seeds in brain extracts from PD and MSA patients. Both soluble and insoluble fractions of MSA extracts had robust seeding activity, whereas only the insoluble fractions of PD extracts displayed seeding activity. The morphology of MSA-seeded inclusions differed from PD-seeded inclusions. These differences persisted upon propagation of aggregation to second-generation biosensor cells. We conclude that PD and MSA feature α-syn conformers with very distinct biochemical properties that can be transmitted to α-syn monomers in a cell system. These findings are consistent with the idea that distinct α-syn strains underlie PD and MSA and offer possible directions for synucleinopathy diagnosis.

Chalcones and Five-Membered Heterocyclic Isosteres Bind to Alpha Synuclein Fibrils in Vitro.

A series of chalcone and heterocyclic isosteres, in which the enone moiety was replaced with an isoxazole and pyrazole ring system, was synthesized and their affinities for alpha synuclein (Asyn), amyloid beta (Aß), and tau fibrils were measured in vitro. The compounds were found to have a modest affinity and selectivity for Asyn versus Aß fibrils and low affinity for tau fibrils. Insertion of a double bond to increase the extendable surface area resulted in an increase in affinity and improvement in selectivity for Asyn versus Aß and tau fibrils. The results of this study indicate that compound 11 is a secondary lead compound for structure-activity relationship studies aimed at identifying a suitable compound for positron emission tomography-imaging studies of insoluble Asyn aggregates in Parkinson's disease.

Alpha Synuclein Fibrils Contain Multiple Binding Sites for Small Molecules.

The fibrillary aggregation of the protein alpha synuclein (Asyn) is a hallmark of Parkinson's disease, and the identification of small molecule binding sites on fibrils is essential to the development of diagnostic imaging probes. A series of molecular modeling, photoaffinity labeling, mass spectrometry, and radioligand binding studies were conducted on Asyn fibrils. The results of these studies revealed the presence of three different binding sites within fibrillar Asyn capable of binding small molecules with moderate to high affinity. A knowledge of the amino acid residues in these binding sites will be important in the design of high affinity probes capable of imaging fibrillary species of Asyn.

Resonance assignments of an α-synuclein fibril prepared in Tris buffer at moderate ionic strength.

Fibrils of the protein α-synuclein (α-syn) are implicated in the pathogenesis of Parkinson's disease and related neurodegenerative disorders. We have reported a high-resolution structure (PDB 2N0A) of an α-syn fibril form prepared by in vitro incubation of monomeric protein in 50 mM sodium phosphate buffer pH 7.4 with 0.1 mM EDTA and 0.01% sodium azide. In parallel with this structure determination, ongoing studies of small molecule ligands binding to α-syn fibrils, prepared in 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) buffer, have been in progress, and it is therefore of interest to determine the structural similarity of these forms. Here we report the 13C and 15N resonance assignments for α-syn fibrils prepared with Tris-HCl buffer (pH 7.7 at 37 °C) and 100 mM NaCl. These fibrillization conditions yield a form with fibril core chemical shifts highly similar to those we reported (BMRB 16939) in the course of determining the high-resolution 2N0A structure, with the exception of some small perturbations from T44 to V55, including two sets of peaks observed for residues T44-V48. Additional differences occur in the patterns of observed residues in the primarily unstructured N-terminus. These results demonstrate a common fold of the fibril core for α-syn fibrils prepared in phosphate or Tris-HCl buffer at moderate ionic strength.

Design, synthesis, and in vitro evaluation of quinolinyl analogues for α-synuclein aggregation.

Here we report the synthesis and in vitro evaluation of 25 new quinolinyl analogues for α-synuclein aggregates. Three lead compounds were subsequently labeled with carbon-11 or fluorine-18 to directly assess their potency in a direct radioactive competitive binding assay ng both α-synuclein fibrils and tissue homogenates from Alzheimer's disease (AD) cases. The modest binding affinities of these three radioligands toward α-synuclein were comparable with results from the Thioflavin T fluorescence assay. However, all three ligand also showed modest binding affinity to the AD homogenates and lack selectivity for α-synuclein. The structure-activity relationship data from these 25 analogues will provide useful information for design and synthesis of new compounds for imaging α-synuclein aggregation.

A sensitive assay reveals structural requirements for α-synuclein fibril growth.

The accumulation of α-synuclein (α-syn) fibrils in neuronal inclusions is the defining pathological process in Parkinson's disease (PD). A pathogenic role for α-syn fibril accumulation is supported by the identification of dominantly inherited α-syn (SNCA) gene mutations in rare cases of familial PD. Fibril formation involves a spontaneous nucleation event in which soluble α-syn monomers associate to form seeds, followed by fibril growth during which monomeric α-syn molecules sequentially associate with existing seeds. To better investigate this process, we developed sensitive assays that use the fluorescein arsenical dye FlAsH (fluorescein arsenical hairpin binder) to detect soluble oligomers and mature fibrils formed from recombinant α-syn protein containing an N-terminal bicysteine tag (C2-α-syn). Using seed growth by monomer association (SeGMA) assays to measure fibril growth over 3 h in the presence of C2-α-syn monomer, we observed that some familial PD-associated α-syn mutations (i.e. H50Q and A53T) greatly increased growth rates, whereas others (E46K, A30P, and G51D) decreased growth rates. Experiments with wild-type seeds extended by mutant monomer and vice versa revealed that single-amino acid differences between seed and monomer proteins consistently decreased growth rates. These results demonstrate that α-syn monomer association during fibril growth is a highly ordered process that can be disrupted by misalignment of individual amino acids and that only a subset of familial-PD mutations causes fibril accumulation through increased fibril growth rates. The SeGMA assays reported herein can be utilized to further elucidate structural requirements of α-syn fibril growth and to identify growth inhibitors as a potential therapeutic approach in PD.

Fluselenamyl: A Novel Benzoselenazole Derivative for PET Detection of Amyloid Plaques (Aß) in Alzheimer's Disease.

Fluselenamyl (5), a novel planar benzoselenazole shows traits desirable of enabling noninvasive imaging of Aß pathophysiology in vivo; labeling of both diffuse (an earlier manifestation of neuritic plaques) and fibrillar plaques in Alzheimer's disease (AD) brain sections, and remarkable specificity for mapping Aß compared with biomarker proteins of other neurodegenerative diseases. Employing AD homogenates, [18F]-9, a PET tracer demonstrates superior (2-10 fold higher) binding affinity than approved FDA tracers, while also indicating binding to high affinity site on Aß plaques. Pharmacokinetic studies indicate high initial influx of [18F]-9 in normal mice brains accompanied by rapid clearance in the absence of targeted plaques. Following incubation in human serum, [18F]-9 indicates presence of parental compound up to 3h thus indicating its stability. Furthermore, in vitro autoradiography studies of [18F]-9 with AD brain tissue sections and ex vivo autoradiography studies in transgenic mouse brain sections show cortical Aß binding, and a fair correlation with Aß immunostaining. Finally, multiphoton- and microPET/CT imaging indicate its ability to penetrate brain and label parenchymal plaques in transgenic mice. Following further validation of its performance in other AD rodent models and nonhuman primates, Fluselenamyl could offer a platform technology for monitoring earliest stages of Aß pathophysiology in vivo.

Design, Synthesis, and Characterization of 3-(Benzylidene)indolin-2-one Derivatives as Ligands for α-Synuclein Fibrils.

A series of 3-(benzylidine)indolin-2-one derivatives were synthesized and evaluated for their in vitro binding to alpha synuclein (α-syn), beta amyloid (Aß), and tau fibrils. Compounds with a single double bond in the 3-position had only a modest affinity for α-syn and no selectivity for α-syn versus Aß or tau fibrils. Homologation to the corresponding diene analogues yielded a mixture of Z,E and E,E isomers; substitution of the indoline nitrogen with an N-benzyl group resulted in increased binding to α-syn and reasonable selectivity for α-syn versus Aß and tau. Introduction of a para-nitro group into the benzene ring of the diene enabled separation of the Z,E and E,E isomers and led to the identification of the Z,E configuration as the more active regioisomer. The data described here provide key structural information in the design of probes which bind preferentially to α-syn versus Aß or tau fibrils.

Synthesis, characterization, and preclinical validation of a PET radiopharmaceutical for interrogating Aß (ß-amyloid) plaques in Alzheimer's disease.

BACKGROUND: PET radiopharmaceuticals capable of imaging ß-amyloid (Aß) plaque burden in the brain could offer highly valuable diagnostic tools for clinical studies of Alzheimer's disease. To further supplement existing armamentarium of FDA-approved agents as well as those under development, and to correlate multiphoton-imaging data reported earlier, herein, we describe preclinical validation of a PET tracer. METHODS: A novel PET radiopharmaceutical ((18)F-7B) was synthesized and characterized. To assess its affinity for Aß, binding assays with Aß1-42 fibrils, Alzheimer's disease (AD) homogenates, and autoradiography studies and their IHC correlations were performed. For assessing its overall pharmacokinetic profiles in general and its ability to cross the blood-brain barrier (BBB) in particular, biodistribution studies in normal mice were performed. Finally, for evaluating potential for (18)F-7B to serve as a targeted Aß probe, the microPET/CT imaging was performed in age-matched amyloid precursor protein/presenilin-1 (APP/PS1) mice and wild-type (WT) counterparts. RESULTS: The radiotracer (18)F-7B shows saturable binding to autopsy-confirmed AD homogenates (K d = 17.7 nM) and Aß1-42 fibrils (K d = 61 nM). Preliminary autoradiography studies show binding of (18)F-7B to cortical Aß plaques in autopsy-confirmed AD tissue sections, inhibition of that binding by unlabeled counterpart 7A-indicating specificity, and a good correlation of tracer binding with Aß immunostaining. The agent indicates high initial penetration into brains (7.23 ± 0.47%ID/g; 5 min) of normal mice, thus indicating a 5-min/120-min brain uptake clearance ratio of 4.7, a benchmark value (>4) consistent with the ability of agents to traverse the BBB to enable PET brain imaging. Additionally, (18)F-7B demonstrates the presence of parental species in human serum. Preliminary microPET/CT imaging demonstrates significantly higher retention of (18)F-7B in brains of transgenic mice compared with their WT counterparts, consistent with expected binding of the radiotracer to Aß plaques, present in APP/PS1 mice, compared with their age-matched WT counterparts lacking those Aß aggregates. CONCLUSIONS: These data offer a platform scaffold conducive to further optimization for developing new PET tracers to study Aß pathophysiology in vitro and in vivo.