Molecular imaging of butyrylcholinesterase associated with amyloid-β plaques distinguishes 5XFAD from wild-type mice

Molecular imaging of butyrylcholinesterase associated with amyloid-β plaques distinguishes 5XFAD from wild-type mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular imaging of butyrylcholinesterase associated with amyloid-β plaques distinguishes 5XFAD from wild-type mice G. Andrew Reid, Drew R. DeBay, Ian R. Macdonald, Antoun Bou Laouz, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7491155/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Purpose Diagnosis of Alzheimer’s disease (AD) requires symptoms of dementia and accumulation of amyloid-β (Aβ) and tau in the brain. Molecular imaging of Aβ or tau in AD, though informative, is complicated by the finding that similar changes are found in brains of ~ 30% of cognitively normal older individuals. Butyrylcholinesterase (BChE), normally present in low levels in the cerebral cortex, is found in high levels associated with Aβ plaques in AD. When associated with this pathological structure, the biochemical properties of BChE are altered. The aim of the present study was to determine if [ 18 F]1-Methyl-4-piperidinyl p-fluorobenzoate ([ 18 F]BMP) can image BChE associated with Aβ in 5XFAD mouse model of AD and distinguish it from its wild-type (WT) counterpart. Procedures [ 18 F]BMP was synthesized and evaluated in wild-type (WT), 5XFAD and BChE knock-out (BChE-KO) mouse models for in vivo dynamic PET imaging of BChE. Time-activity curves were generated and [ 18 F]BMP clearance parameters were determined. Brain, liver and urine homogenates were evaluated for [ 18 F]BMP and its metabolites. Ex vivo autoradiography mapped the distribution of [ 18 F]BMP brain retention. Results In vivo PET imaging following injection of [ 18 F]BMP demonstrated significantly greater brain retention of activity in the 5XFAD mouse model compared to WT, while BChE-KO mice mirrored WT levels. Metabolite analysis confirmed [ 18 F]BMP was metabolized in the periphery but survived in sufficient quantity to enter the brain. Ex vivo autoradiography showed [ 18 F]BMP retention in the 5XFAD mouse brain where BChE-associated Aβ plaques were prominent. Conclusions These results demonstrate that PET imaging of BChE-associated Aβ plaques is feasible, offering an avenue to evaluate role(s) of BChE in AD pathogenesis, progression and complement the existing AD biomarker framework. Alzheimer’s disease PET imaging ex vivo autoradiography Aβ biomarker acetylcholinesterase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Alzheimer’s disease (AD) presents clinically with dementia and is defined pathologically by the presence of brain amyloid-β (Aβ) plaques, tau neurofibrillary tangles and extensive loss of neurons [ 1 ]. These pathological changes can be detected using molecular and anatomical neuroimaging, as well as cerebrospinal fluid and blood-based biomarkers [ 2 ]. While these biomarkers have improved AD pathology detection in vivo , there remain challenges. AD pathology in cognitively normal older individuals poses a clinical uncertainty, as biomarker Aβ or tau positivity are insufficient to predict the development of symptomatic AD [ 3 ]. Thus, a multimodal approach, using various biomarkers, is likely required to improve AD diagnosis and disease progression monitoring [ 4 ]. Alongside the accumulation of Aβ and tau, changes to the cholinergic system are an important feature in AD progression [ 5 , 6 ]. This includes the cholinergic enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which undergo changes in their levels, distribution and kinetic properties. AChE levels have been shown in vitro to be decreased in AD while that of BChE either remains the same or increases [ 7 , 8 ]. Histochemical studies have shown a change in distribution, with a loss of AChE-associated normal neural elements and an increased association of AChE and BChE with Aβ plaques [ 8 , 9 ]. When associated with these pathological structures, their properties such as inhibitor sensitivity, substrate affinity and optimum pH are altered [ 8 , 10 ], likely due to the conformational changes [ 11 , 12 ]. In human brains at autopsy, BChE is a highly sensitive and specific biomarker that distinguishes AD pathology from similar pathology present in cognitively normal brains [ 13 ]. Furthermore, BChE specifically associates with AD pathology and not with pathology associated with other dementia syndromes such as dementia with Lewy bodies, corticobasal syndrome or frontotemporal dementia [ 13 ]. These changes suggest that targeting BChE for molecular imaging can contribute to a multimodal approach of diagnosing AD and monitoring disease progression that will augment current biomarker approaches. Several radioligands have been developed targeting AChE and BChE in the brain. In vivo studies using N -[ 11 C] methylpiperidin-4-yl acetate ([ 11 C]MP4A) and N -[ 11 C] methylpiperidin-4-yl propionate ([ 11 C]MP4P) have effectively measured AChE-associated neurons and neuropil in normal brain and in neurodegenerative disorders [ 14 ] but not AChE associated with AD pathology. Similarly, the BChE-specific PET ligand, 1-[ 11 C] methylpiperidin-4-yl butyrate ([ 11 C]MP4B), did not demonstrate BChE activity levels in the AD cortex [ 15 , 16 ] as predicted by in vitro studies [ 7 , 8 ]. In contrast, the BChE-specific SPECT ligand 1-methyl-4-piperidinyl p- [ 123 I]iodobenzoate [ 17 , 18 ] was retained at higher levels in the brains of an amyloid mouse model (5XFAD) compared to a wild-type (WT) counterpart [ 19 ]. A PET analogue of this ligand,1-methyl-4-piperidinyl p- [ 18 F]fluorobenzoate ([ 18 F]BMP), was previously evaluated as a potential AChE imaging agent in WT mice and determined to be unsuitable [ 20 ]. However, additional studies confirmed 1-methyl-4-piperidinyl p- fluorobenzoate (BMP) was a substrate for BChE, not AChE [ 18 ]. Considering the results of its SPECT analogue, we examined [ 18 F]BMP as a BChE imaging agent in the 5XFAD mouse. The present study aimed to assess if [ 18 F]BMP in vivo PET imaging can distinguish 5XFAD mice from WT by engaging BChE-associated with Aβ plaques. The 5XFAD mouse is an aggressive model of familial amyloidosis [ 21 ] that develops Aβ plaques with BChE activity [ 22 ] similar to AD, making this model suitable to evaluate BChE PET ligands for identifying AD pathology. In addition, mice lacking BChE (BChE-KO) [ 23 ] were evaluated as additional controls to determine non-specific/off-target uptake. Metabolite analysis and ex vivo autoradiography were completed to confirm target engagement in PET studies. Materials & Methods All experiments were approved by the Dalhousie University Radiation Safety Committee, the Canadian Nuclear Safety Commission (07154-2-17.10) and Dalhousie University Animal Care Committee (24–037). Mice were cared for in accordance with Canadian Council on Animal Care guidelines. Chemical synthesis [ 18 F]BMP ( 1 ), BMP ( 2 ), 1-methylpiperidin-4-yl p -nitrobenzoate ( 3 ) and [ 18 F]4-fluorobenzoic acid ([ 18 F]FBA, 4 ) were synthesized (Supplementary Scheme 1) as described previously [ 20 , 24 ]. Details and analytical data are available in the Supplementary Material. Dynamic PET and MR Imaging Dynamic PET imaging, following lateral tail vein injection of [ 18 F]BMP, was completed in WT (three ♀ and four ♂), 5XFAD (three ♀ and three ♂) and BChE-KO (three ♀ and three ♂) mice with an average age of 13.3 ± 0.8 months (Supplementary Table 1). PET imaging following injection of [ 18 F]FBA was completed in a 13.6-month-old male, 5XFAD mouse. Details on mouse strain and PET/MR image methods are provided in the Supplementary Material. Dynamic PET Data Analysis Dynamic PET data were analyzed using PMOD (PMOD Technologies, Zurich, Switzerland, version 4.106) to evaluate and compare uptake and clearance of [ 18 F]BMP and [ 18 F]FBA in mice. Reconstructed PET DICOM files were opened in Amide (version 1.0.4) and exported as DIMCOM 3.0 via (X)MedCon files prior to being loaded into PMOD. For anatomical reference, PET and corresponding MRI data were fused using a rigid body transformation and visually inspected to ensure goodness of fit. Calibrated PET data (MBq/mm 3 ) were converted into % injected dose per ml (%ID/ml) and %ID. Volumes of interest (VOIs) for the lungs, heart, liver and kidneys were generated using standardized ellipsoids placed within each organ. A standardized whole brain VOI for examining biodistribution was generated using the PMOD iso-contouring tool. All other analysis of brain data used the Ma-Benveniste-Mirrione template atlas [ 25 ]. Time-activity curve (TAC) data generated for each mouse was exported from PMOD and analyzed in Excel or Prism (Supplementary Material - Statistical Analysis section). Metabolite Analysis In vivo analysis of [ 18 F]BMP was performed 20 min after injection to assess the ligand and its metabolite(s) in the periphery and brain, as described previously (20). Details summarized in the Supplementary Material. Ex Vivo Autoradiography Ex vivo autoradiography, following injection of [ 18 F]BMP, was completed in WT (three ♀ and five ♂), 5XFAD (four ♀ and five ♂) and BChE-KO (one ♂) mice (Supplementary Table 2). A 20 min survival period was selected based on PET TACs. Details available in the Supplementary Material. Butyrylcholinesterase Histochemistry BChE histochemistry was performed on brain tissue sections from all experimental mice as described previously [ 18 ]. Details available in the Supplementary Material. Results Chemical Synthesis Radiosynthesis of [ 18 F]BMP ( 1 ) provided a decay corrected radiochemical yield of 30%, radiochemical purity of 97% (Supplementary Fig. 1) and a molar activity of 12.1 GBq/mmol at the end of synthesis, in line with a previous report [ 20 ]. The HPLC retention time of synthesized [ 18 F]BMP was consistent with BMP ( 2 ) (Supplementary Fig. 2), confirming the identity. BMP ( 2 ) was synthesized in 86% yield with 98% purity (Supplementary Figs. 3–11). 1-Methylpiperidin-4-yl p -nitrobenzoate ( 3 ) yielded 64% with 96% purity (Supplementary Figs. 12–20). Spectroscopic data for compounds 2 and 3 (Supplementary Scheme 1) were consistent with their structure. [ 18 F]FBA ( 4 ) was synthesized by base hydrolysis of [ 18 F]BMP, yielding 24% with 98% radiochemical purity (Supplementary Figs. 21–22). Dynamic PET Data Analysis Following injection of [ 18 F]BMP, its distribution was characterized in mice using dynamic PET imaging. Immediate time points demonstrated ligand perfusion of the inferior vena cava, heart and lungs followed by the brain, liver and, finally, kidneys. Robust brain uptake indicated [ 18 F]BMP effectively crossed the blood-brain barrier (BBB). Brain, heart, lungs, liver and kidneys VOIs (Fig. 1 A) were used to generate biodistribution TACs (Fig. 1 B) from data standardized to percent of the injected dose per ml (%ID/ml). Heart and lung uptake was rapid, while that in the liver and kidneys was comparatively delayed. Washout of activity from the heart and lungs was rapid while clearance in the liver was slower. Uptake and washout in the kidneys varied between mice whereby in the majority, a gradual washout was observed over time while in other mice, uptake continued over the duration of the scan. Averaged TAC data from PET scans illustrate the uptake and clearance of [ 18 F]BMP in the brains of WT, 5XFAD and BChE-KO mice (Fig. 2 ). Distinct separation between the 5XFAD TAC and both WT and BChE-KO curves were apparent, whereby [ 18 F]BMP clearance from the brains of female and male 5XFAD mice appeared to be slower than their WT counterparts (Fig. 2 ). These differences were typically apparent 10 min after injection of [ 18 F]BMP and continued until 50 min (Fig. 3 ). The TACs for BChE-KO mice appeared comparable to WT mice (Figs. 2 and 3 ). Statistical analysis of [ 18 F]BMP dynamic PET data demonstrated several differences between strains and sex. Although the average C max did not differ between strains it was higher in female mice (1.5%ID) compared to male (1.2%ID, *** p < 0.001). Consequently, sexes were analyzed separately. The %ID in the brains of 5XFAD mice were significantly higher at timepoints throughout the PET scan (Supplementary Tables 3 and 4). Compared to WT counterparts, the %ID in the brains of female 5XFAD mice was 43.8% higher at 20 mins and 97.8% at 50 mins with significant differences at 10 and 20 mins post injection. In male 5XFAD mice, the brain %ID was increased 27.6% at 20 min and 67.5% at 50 mins compared to WT counterparts with significant differences at 10, 20, 30, 40 and 50 mins. The rates of clearance or half-life of ligand activity (t 1/2 clearance , Table 1 ) were significantly longer in 5XFAD mice relative to WT counterparts (Fig. 4 , Supplementary Table 5). Compared to WT mice, the t 1/2 clearance was 33% higher in female 5XFAD mice and 39% higher in males. Table 1 Average data and standard deviation of the t 1/2 clearance derived from an exponential decay curve model of 1-methyl-4-piperidinyl p -fluorobenzoate ([ 18 F]BMP) dynamic PET data in wild-type (WT), 5XFAD and butyrylcholinesterase knockout (BChE-KO) mice. The average R 2 with standard deviation is shown demonstrating the goodness of fit of the modelled curve. t 1/2 clearance (min) R 2 Female WT 5.46 ± 0.37 0.99 ± < 0.001 5XFAD 7.28 ± 0.56 0.99 ± 0.002 BChE-KO 5.41 ± 0.46 0.99 ± < .01 Male WT 5.41 ± 0.45 0.99 ± < 0.001 5XFAD 7.51 ± 0.29 0.99 ± 0.002 BChE-KO 4.89 ± 0.22 0.99 ± 0.001 PET imaging data in BChE-KO mice assessed off-target/non-specific interactions of [ 18 F]BMP in the brain. Uptake, clearance (Table 1 ) and the %ID in the whole brain VOI (Supplementary Table 3) of male and female BChE-KO mice closely matched WT counterparts (Figs. 2 , 3 , 4 ). Statistical analysis showed no significant differences in the %ID in the brain at any timepoint (Supplementary Table 4) or t 1/2clearance (Supplementary Table 5). PET imaging of a male 5XFAD mouse injected with [ 18 F]FBA, a radioactive [ 18 F]BMP metabolite, showed limited BBB crossing and relatively quick washout from the brain (Supplementary Fig. 23). Compared to the average values of [ 18 F]BMP in male 5XFAD mice, whole brain C max of [ 18 F]FBA was 3.7x lower while the brain clearance (t 1/2 clearance ) was 21% faster. Peripheral biodistribution was similar for both ligands. Metabolite Analysis Brain, liver and urine were analyzed for parent compound and metabolites 20 min after [ 18 F]BMP injection in female WT, 5XFAD and BChE-KO mice. HPLC analysis of 5XFAD brain isolate showed clear presence of [ 18 F]BMP, while WT and BChE-KO samples had lower activity and no clear [ 18 F]BMP peaks. Liver isolate samples from all strains contained [ 18 F]BMP and radioactive metabolites. Urine from all strains showed high activity from metabolites, with minor [ 18 F]BMP detected. See Supplementary Fig. 24 for HPLC data. Ex vivo autoradiography Ex vivo autoradiography with [ 18 F]BMP showed clear differences between WT and 5XFAD mice. In WT brains, activity was mainly within white matter (Fig. 5 ) while the cortical grey matter, striatum, thalamus, hippocampus and brainstem showed less and remained uniform. Notably, brain regions with higher levels of BChE associated with neurons and neuropil [ 26 ] were not associated with increased retention. In contrast, 5XFAD mouse brain showed higher activity in the cortex, amygdala and the hippocampal formation, particularly the subiculum, regions with BChE-associated Aβ plaques (Fig. 5 ). Uptake in the 5XFAD hypothalamus, striatum, brainstem and cerebellum appeared generally consistent with WT counterparts. Ex vivo autoradiography in a 5.5-month-old 5XFAD female showed a similar pattern of retention compared to older mice (Supplementary Fig. 25). Additional data from male and female, WT and 5XFAD mice are provided in Fig. 25 of the Supplementary Material. Ex vivo autoradiography in the BChE-KO mouse appeared consistent with that in WT (Supplementary Fig. 25). Butyrylcholinesterase Histochemistry There was robust tissue staining of BChE enzymatic activity in the brain sections of 5XFAD and WT mice from PET imaging studies (Fig. 5 ). In WT, staining was associated with white matter, glia and select populations of neurons, as shown previously [ 26 ]. Staining of white matter, neuropil and neurons was also seen in 5XFAD brain sections but there was also staining associated with Aβ plaques (Fig. 5 ), as shown previously [ 19 , 22 ]. There was no staining of BChE activity in the brain sections from BChE-KO mice. Staining of brain tissue sections of mice from ex vivo autoradiography studies resulted in intense staining artefact associated with cell nuclei and poor staining of BChE activity associated with neurons, neuropil and Aβ plaques. Consequently, brain sections from mice used in PET imaging were compared to autoradiography shown in Fig. 5 . Discussion The current study evaluated 1-methyl-4-piperidinyl p- [ 18 F]fluorobenzoate ([ 18 F]BMP) in WT, 5XFAD and BChE-KO mice for brain uptake and localization to BChE associated with Aβ plaques and normal neural elements (i.e. neurons, glia, neuropil, white matter) as well as off-target/non-specific interactions. [ 18 F]BMP was synthesized efficiently and remained stable for PET imaging, metabolite and autoradiography studies. Despite peripheral metabolism, [ 18 F]BMP crossed the BBB sufficiently for brain visualization (Figs. 1 , 2 , 3 ). This contrasted with its metabolite [ 18 F]FBA, which showed poor BBB penetration and faster brain clearance (Supplementary Fig. 23). Brain clearance of [ 18 F]BMP was significantly slower in 5XFAD mice compared to its WT counterpart, illustrated by the TACs (Fig. 2 ), the %ID remaining in the brain at specific timepoints (Fig. 3 , Supplementary Tables 3 and 4) and t 1/2 clearance (Fig. 4 , Table 1 , Supplementary Table 5). In vivo metabolite analysis indicated [ 18 F]BMP was not metabolized in the brain (Supplementary Fig. 24). Ex vivo autoradiography confirmed 5XFAD brain retention of [ 18 F]BMP in regions where BChE was associated with Aβ plaques but not normal neural elements (Fig. 5 , Supplementary Fig. 25). These results indicate [ 18 F]BMP specifically visualizes BChE-associated Aβ plaques and differentiates 5XFAD from WT mice. Cholinesterases (ChEs) associated with Aβ plaques have altered kinetic properties, including pH effects, inhibitor sensitivity and substrate affinities [ 8 , 10 ], that may affect an imaging agent’s ability to accurately reflect ChE distribution in the AD brain. For example, PET imaging with AChE-specific substrates such as [ 11 C]MP4A and [ 11 C]MP4P have successfully shown the distribution of this enzyme associated with neural structures in the normal and diseased brain [ 14 ] but did not recapitulate the distribution of AChE-associated Aβ plaques. Similarly, the BChE ligand [ 11 C]MP4B showed generally decreased BChE levels in AD rather than the higher levels due to BChE-associated Aβ plaques [ 16 ]. In addition, a highly potent BChE inhibitor ligand showed differences between 5XFAD and WT mice in younger age groups but not older, when BChE-associated Aβ plaques levels were higher [ 27 ]. These observations suggest ligand affinity for ChEs bound to normal neural elements and Aβ plaques may differ, possibly due to altered kinetic properties [ 8 , 10 ] resulting from conformational changes [ 11 , 12 ]. Consequently, an imaging agent that accurately recapitulates the levels of ChEs associated with normal neural elements may not interact with and successfully image ChEs associated with Aβ plaques and vice versa . Dynamic PET imaging and ex vivo autoradiography data suggests [ 18 F]BMP may exhibit higher affinity for Aβ plaque bound BChE compared to that associated with normal neural elements. In WT mice, BChE is associated with glia, white matter and a distinct population of neurons [ 26 ]. Consequently, PET imaging of a BChE ligand would be expected to yield differing imaging profiles between WT and BChE-KO mice. However, our analysis of [ 18 F]BMP between these two strains found no significant differences implying limited engagement of [ 18 F]BMP with BChE-associated normal neural elements. These comparable results also suggest that brain retention in these strains may, at least partially, represent non-specific, off-target binding commonly seen with other brain imaging agents, including routinely used clinical amyloid ligands [ 28 ]. Such non-specific interactions are likely also present, to a degree, in the 5XFAD mouse. However, ex vivo autoradiography studies clearly demonstrate the increased retention of [ 18 F]BMP observed in PET imaging studies is due to the localization of activity in regions exhibiting high levels of BChE-associated Aβ plaques, underlying the overall increased ligand retention in this mouse model of AD. The in vivo enzyme kinetic behavior of [ 18 F]BMP may account for its favorable imaging of BChE-associated with Aβ plaques. Our previous histochemical studies evaluating potential ChE imaging agents in brain tissue sections provided insights into their kinetic behavior when interacting with ChEs associated with normal neural elements and Aβ plaques [ 18 , 24 ]. These studies demonstrated that, although in vitro kinetics showed BMP was a BChE-specific substrate with properties of a potential metabolic trapping imaging agent, it was not hydrolyzed well in the brain and may, in fact, engage BChE as an inhibitor [ 18 ]. The current work and previous reports [ 20 ], also indicates [ 18 F]BMP is not hydrolyzed in the brain. These findings suggest [ 18 F]BMP may have a prolonged interaction with BChE, acting as an inhibitor, and function as a molecular imaging agent capable of visualizing BChE associated with Aβ plaques. However, the mechanism of [ 18 F]BMP interaction with BChE-associated Aβ plaques remains unclear. Previous studies have shown other compounds inhibit BChE-associated Aβ plaques by binding to remote allosteric sites of the enzyme [ 29 ]. As [ 18 F]BMP is not hydrolyzed in the brain, this ligand may engage BChE-associated Aβ plaques similarly. The bioavailability of [ 18 F]BMP in WT, 5XFAD and BChE-KO mice appeared comparable. Peripheral metabolism of [ 18 F]BMP in BChE-KO mice matched that in WT and 5XFAD mice suggesting that enzymes other than BChE may be largely responsible for its metabolism in the periphery. Comparable C max values indicate similar parent to metabolite ratio (i.e. [ 18 F]BMP versus [ 18 F]FBA) in the periphery. Dynamic PET imaging showed [ 18 F]FBA, the main metabolite of [ 18 F]BMP, does not sufficiently cross the BBB, resulting in significantly lower uptake and C max . Overall, [ 18 F]BMP brain bioavailability appeared consistent across all strains of mice. Future studies will be required to validate [ 18 F]BMP as a potential AD PET imaging ligand in humans. Regional analysis of brain uptake may clarify regions with intense in vivo uptake (e.g. cerebral cortex) improving sensitivity and specificity for differentiating AD from non-AD brains rather than whole brain uptake in this study. In vivo metabolite analysis showed metabolism in the blood. Consequently, a metabolite correction will be necessary to derive PET kinetic parameters. Although the bioavailability of [ 18 F]BMP was sufficient for brain imaging, determining the plasma free fraction may yield useful information as well. To show the value of [ 18 F]BMP as an AD imaging agent, it will be necessary to test in mice at earlier age points than that used in this study. However, ex vivo autoradiography in a 5.5-month-old female 5XFAD mouse (Supplementary Fig. 25) showed retention comparable to older mice, suggesting potential AD pathology detection at earlier stages. Conclusion The current work provides a proof-of-principle that the [ 18 F]BMP PET ligand can distinguish the 5XFAD amyloid mouse model from its WT counterpart by visualizing BChE-associated Aβ plaques. Molecular imaging of BChE-associated AD pathology may enhance the multi-modal approach of AD diagnosis, distinguishing cognitively normal individuals with plaques from those with AD, as well as present an avenue for monitoring of disease progression. Declarations Author Contributions : G. Andrew Reid: Contributed to completion of experiments, analysis and interpretation of data as well as drafting, revision and approving final content of manuscript. Drew R. DeBay: Contributed to conception and design, analysis and interpretation of data as well as drafting, revision and approving final content of manuscript. Ian R. Macdonald: Contributed to the analysis and interpretation of the data as well as drafting, revision and approving the final content of the manuscript. Antoun Bou Laouz: Contributed to ensuring availability of radioactive materials from the cyclotron, discussion about purity of radioactive raw material, reviewing information and data related to the radioactive materials, revising the manuscript and approving the final version of the manuscript. Ian R. Pottie: Contributed to conception and design, funding acquisition, aquiring, analysis and interpretation of data and drafting, revision and approval of final content of manuscript. Sultan Darvesh: Project conceptualization, funding acquisition, supervision, data curation, validation and analysis, resources, writing, review, editing and approval of final version of the manuscript. Acknowledgements: We would like to thank Dr. John Fisk and Meghan Cash for critically reading this manuscript and Hillary Maillet, Meagan Laffin, Emily MacAulay, Danielle Aucoin, Matt Sawler, Jessica George, Deanna Burns, K. Brewer, C. Davis for their technical assistance. Funding : This research was supported by the Canadian Institutes of Health Research (MOP-82798, RNS- 117795, MOP-119343, PJT – 153319), the Canadian Foundation for Innovation (Grant No. 37854), the Dalhousie Medical Research Foundation (DMRF Chemists, DMRF Clare Durland Fund in Alzheimer’s Disease Research; DMRF Research Grant – Robert and Barbara Pickett; DMRF Gillian’s Hope for MS Research Fund, Mrs. Sadie MacLeod through the Dalhousie Medical Research Foundation Adopt-a-Researcher program), and the Dalhousie University Endowed Irene MacDonald Sobey Endowed Chair in Curative Approaches to Alzheimer’s Disease. Conflicts of Interest : Sultan Darvesh is a scientific co-founder and stockholder in Treventis Corporation. Sultan Darvesh, Ian R. Pottie and Ian R. Macdonald are listed as co-inventors on patents assigned to Treventis Corporation. No other authors have financial disclosures or conflicts of interest with this submission. 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Aging 17(3):822–850. 10.18632/aging.206227 Supplementary Files ElectronicSupportingMaterial.pdf SupplementaryScheme1.tif GA.png Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revisions 16 Dec, 2025 Reviewers agreed at journal 27 Nov, 2025 Reviewers invited by journal 11 Nov, 2025 Editor assigned by journal 01 Sep, 2025 First submitted to journal 29 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7491155","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":543455103,"identity":"3d4f6c9a-f28b-46cc-8b51-971549196e5c","order_by":0,"name":"G. Andrew Reid","email":"","orcid":"","institution":"Dalhousie University","correspondingAuthor":false,"prefix":"","firstName":"G.","middleName":"Andrew","lastName":"Reid","suffix":""},{"id":543455104,"identity":"186ffac5-d53f-431a-ab38-d8203299cbec","order_by":1,"name":"Drew R. 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09:15:20","extension":"html","order_by":58,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124275,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7491155/v1/1b19d388187bc311a514e860.html"},{"id":96556139,"identity":"a2ed4d92-16d0-47df-a4aa-9935efa001b4","added_by":"auto","created_at":"2025-11-23 11:45:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":221794,"visible":true,"origin":"","legend":"\u003cp\u003eBiodistribution of activity following injection of 1-methyl-4-piperidinyl \u003cem\u003ep\u003c/em\u003e-fluorobenzoate ([\u003csup\u003e18\u003c/sup\u003eF]BMP)\u003cstrong\u003e.\u0026nbsp; \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) PET images in the coronal, sagittal and axial planes (left to right) showing the average % injected dose per ml (%ID/ml) in the 2 min following injection of [\u003csup\u003e18\u003c/sup\u003eF]BMP in a representative male 5XFAD mouse.\u0026nbsp; The volumes of interest (VOIs) for the brain, heart, lungs, liver and kidneys are outlined.\u0026nbsp; Color scale 0-5 %ID/ml.\u0026nbsp; (\u003cstrong\u003eB\u003c/strong\u003e) Biodistribution time-activity curves for VOIs of the mouse shown in \u003cstrong\u003eA\u003c/strong\u003e illustrating the rapid uptake and washout in the heart and lungs, rapid uptake and slower washout in the brain, liver and kidneys.\u0026nbsp; CD = caudal, CR = cranial, D = dorsal, LE = left, RT = right, V = ventral.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7491155/v1/01637db4b537e0ea5bb6068e.png"},{"id":96556136,"identity":"edb1dc0e-dacd-4f8e-942b-d9d07c378ddc","added_by":"auto","created_at":"2025-11-23 11:45:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97480,"visible":true,"origin":"","legend":"\u003cp\u003eTime-activity curves derived from dynamic PET imaging showing the average % injected dose in the brain following injection of 1-methyl-4-piperidinyl \u003cem\u003ep\u003c/em\u003e-fluorobenzoate ([\u003csup\u003e18\u003c/sup\u003eF]BMP)\u003cstrong\u003e \u003c/strong\u003ein female and male, wild-type (WT), 5XFAD and butyrylcholinesterase knockout (BChE-KO) mice.\u0026nbsp; Note the increased retention of activity in both female and male 5XFAD mice compared to their respective WT counterparts as well as the similarity between WT and BChE-KO mice.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7491155/v1/0fc28a944d39dfb79609d69f.png"},{"id":96556132,"identity":"21005bce-2399-478c-a334-b17b1618734a","added_by":"auto","created_at":"2025-11-23 11:45:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":907815,"visible":true,"origin":"","legend":"\u003cp\u003ePET imaging series showing uptake and retention over 50 minutes, in the axial plane, following injection of 1-methyl-4-piperidinyl \u003cem\u003ep\u003c/em\u003e-fluorobenzoate ([\u003csup\u003e18\u003c/sup\u003eF]BMP)\u003cstrong\u003e \u003c/strong\u003ein a female wild-type (WT), 5XFAD and butyrylcholinesterase knockout\u0026nbsp; (BChE-KO) mouse.\u0026nbsp; Each time frame (0.25, 5.25, 10.25, 20.25, 30.25, 40.25 and 50.25 min) illustrates the average % injected dose/ml (%ID/ml) over a 30 sec period.\u0026nbsp; PET images were co-registered to a brain MRI for anatomical reference (first column).\u0026nbsp; In all strains examined, [\u003csup\u003e18\u003c/sup\u003eF]BMP crossed the blood-brain barrier in a comparable degree.\u0026nbsp; Note the greater retention in the 5XFAD mouse compared to WT following washout.\u0026nbsp; Uptake and retention in the Harderian glands (HG) appeared relatively consistent between strains.\u0026nbsp; Color scale 0-5 %ID/ml. CD = caudal, CR = cranial, LE = left, RT = right.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7491155/v1/8ef38ef00353bb374287663d.png"},{"id":96556140,"identity":"7be4c081-d448-4a19-beb3-f90a1b38cd91","added_by":"auto","created_at":"2025-11-23 11:45:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":153748,"visible":true,"origin":"","legend":"\u003cp\u003eBox and whisker plot showing the whole brain half-life of 1-methyl-4-piperidinyl \u003cem\u003ep\u003c/em\u003e-fluorobenzoate ([\u003csup\u003e18\u003c/sup\u003eF]BMP) activity (t\u003csub\u003e1/2 clearance\u003c/sub\u003e) in female and male, wild-type (WT), 5XFAD and butyrylcholinesterase knockout (BChE-KO) mice.\u0026nbsp; Note, the t\u003csub\u003e1/2 clearance\u003c/sub\u003e is significantly larger in 5XFAD mice.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7491155/v1/f35a0c8c17198fd3719a1ff9.png"},{"id":96604962,"identity":"f9ad0094-e60c-4eb7-8044-2c3a9bffffa1","added_by":"auto","created_at":"2025-11-24 09:16:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":775774,"visible":true,"origin":"","legend":"\u003cp\u003eBrain sections from female wild-type (WT) (columns \u003cstrong\u003eA\u003c/strong\u003e-\u003cstrong\u003eC\u003c/strong\u003e) and 5XFAD mice (columns \u003cstrong\u003eD\u003c/strong\u003e-\u003cstrong\u003eF\u003c/strong\u003e) showing low power photomicrographs of BChE histochemistry at pH 6.8 (\u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eE\u003c/strong\u003e), insets showing staining in the hippocampal formation (HF), amygdala (Am) and cortex (Cx) at a higher magnification (\u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003e) and \u003cem\u003eex vivo\u003c/em\u003e autoradiography following injection of 1-methyl-4-piperidinyl \u003cem\u003ep\u003c/em\u003e-fluorobenzoate ([\u003csup\u003e18\u003c/sup\u003eF]BMP) (\u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e).\u0026nbsp; In WT mouse brain, BChE staining is associated with white matter, glia and neurons (\u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e).\u0026nbsp; In comparable sections, \u003cem\u003eex vivo\u003c/em\u003e autoradiography showed limited retention of activity, mainly associated with white matter (\u003cstrong\u003eC\u003c/strong\u003e).\u0026nbsp; 5XFAD brain sections, there was increased BChE staining associated with plaques in regions including the HF, Am and Cx (\u003cstrong\u003eF\u003c/strong\u003e) which corresponded to areas of increased retention of activity observed with \u003cem\u003eex vivo\u003c/em\u003e autoradiography (\u003cstrong\u003eD\u003c/strong\u003e).\u0026nbsp; Scale bar = 1 mm, inset = 200 µm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7491155/v1/6d71edbcc5ccfc6c23972d4e.png"},{"id":97248997,"identity":"48d718d3-faff-4908-b779-eec736a31ae9","added_by":"auto","created_at":"2025-12-02 13:09:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3141486,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7491155/v1/c8f8dd56-60dd-44ef-a344-bee203b261a2.pdf"},{"id":96556133,"identity":"8ce6565d-291f-427d-87b0-01a94fc1cae1","added_by":"auto","created_at":"2025-11-23 11:45:21","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3033566,"visible":true,"origin":"","legend":"","description":"","filename":"ElectronicSupportingMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7491155/v1/6b7b6f2b1c2f184233315b57.pdf"},{"id":96556150,"identity":"438a77d1-0fe6-4d7b-b7fb-0e52c424aba3","added_by":"auto","created_at":"2025-11-23 11:45:22","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3748936,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryScheme1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7491155/v1/9052143264d8138fdb23ce21.tif"},{"id":96556154,"identity":"2f4e7080-0378-4cc7-ac63-ae3369c6eb49","added_by":"auto","created_at":"2025-11-23 11:45:22","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":653280,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-7491155/v1/774beb98c9edfd96e257551d.png"}],"financialInterests":"","formattedTitle":"Molecular imaging of butyrylcholinesterase associated with amyloid-β plaques distinguishes 5XFAD from wild-type mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) presents clinically with dementia and is defined pathologically by the presence of brain amyloid-β (Aβ) plaques, tau neurofibrillary tangles and extensive loss of neurons [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These pathological changes can be detected using molecular and anatomical neuroimaging, as well as cerebrospinal fluid and blood-based biomarkers [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While these biomarkers have improved AD pathology detection \u003cem\u003ein vivo\u003c/em\u003e, there remain challenges. AD pathology in cognitively normal older individuals poses a clinical uncertainty, as biomarker Aβ or tau positivity are insufficient to predict the development of symptomatic AD [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thus, a multimodal approach, using various biomarkers, is likely required to improve AD diagnosis and disease progression monitoring [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlongside the accumulation of Aβ and tau, changes to the cholinergic system are an important feature in AD progression [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This includes the cholinergic enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which undergo changes in their levels, distribution and kinetic properties. AChE levels have been shown \u003cem\u003ein vitro\u003c/em\u003e to be decreased in AD while that of BChE either remains the same or increases [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Histochemical studies have shown a change in distribution, with a loss of AChE-associated normal neural elements and an increased association of AChE and BChE with Aβ plaques [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. When associated with these pathological structures, their properties such as inhibitor sensitivity, substrate affinity and optimum pH are altered [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], likely due to the conformational changes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In human brains at autopsy, BChE is a highly sensitive and specific biomarker that distinguishes AD pathology from similar pathology present in cognitively normal brains [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, BChE specifically associates with AD pathology and not with pathology associated with other dementia syndromes such as dementia with Lewy bodies, corticobasal syndrome or frontotemporal dementia [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These changes suggest that targeting BChE for molecular imaging can contribute to a multimodal approach of diagnosing AD and monitoring disease progression that will augment current biomarker approaches.\u003c/p\u003e\u003cp\u003eSeveral radioligands have been developed targeting AChE and BChE in the brain. \u003cem\u003eIn vivo\u003c/em\u003e studies using \u003cem\u003eN\u003c/em\u003e-[\u003csup\u003e11\u003c/sup\u003eC] methylpiperidin-4-yl acetate ([\u003csup\u003e11\u003c/sup\u003eC]MP4A) and \u003cem\u003eN\u003c/em\u003e-[\u003csup\u003e11\u003c/sup\u003eC] methylpiperidin-4-yl propionate ([\u003csup\u003e11\u003c/sup\u003eC]MP4P) have effectively measured AChE-associated neurons and neuropil in normal brain and in neurodegenerative disorders [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] but not AChE associated with AD pathology. Similarly, the BChE-specific PET ligand, 1-[\u003csup\u003e11\u003c/sup\u003eC] methylpiperidin-4-yl butyrate ([\u003csup\u003e11\u003c/sup\u003eC]MP4B), did not demonstrate BChE activity levels in the AD cortex [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] as predicted by \u003cem\u003ein vitro\u003c/em\u003e studies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In contrast, the BChE-specific SPECT ligand 1-methyl-4-piperidinyl \u003cem\u003ep-\u003c/em\u003e[\u003csup\u003e123\u003c/sup\u003eI]iodobenzoate [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] was retained at higher levels in the brains of an amyloid mouse model (5XFAD) compared to a wild-type (WT) counterpart [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A PET analogue of this ligand,1-methyl-4-piperidinyl \u003cem\u003ep-\u003c/em\u003e[\u003csup\u003e18\u003c/sup\u003eF]fluorobenzoate ([\u003csup\u003e18\u003c/sup\u003eF]BMP), was previously evaluated as a potential AChE imaging agent in WT mice and determined to be unsuitable [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, additional studies confirmed 1-methyl-4-piperidinyl \u003cem\u003ep-\u003c/em\u003efluorobenzoate (BMP) was a substrate for BChE, not AChE [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Considering the results of its SPECT analogue, we examined [\u003csup\u003e18\u003c/sup\u003eF]BMP as a BChE imaging agent in the 5XFAD mouse.\u003c/p\u003e\u003cp\u003eThe present study aimed to assess if [\u003csup\u003e18\u003c/sup\u003eF]BMP \u003cem\u003ein vivo\u003c/em\u003e PET imaging can distinguish 5XFAD mice from WT by engaging BChE-associated with Aβ plaques. The 5XFAD mouse is an aggressive model of familial amyloidosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] that develops Aβ plaques with BChE activity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] similar to AD, making this model suitable to evaluate BChE PET ligands for identifying AD pathology. In addition, mice lacking BChE (BChE-KO) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] were evaluated as additional controls to determine non-specific/off-target uptake. Metabolite analysis and \u003cem\u003eex vivo\u003c/em\u003e autoradiography were completed to confirm target engagement in PET studies.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cp\u003eAll experiments were approved by the Dalhousie University Radiation Safety Committee, the Canadian Nuclear Safety Commission (07154-2-17.10) and Dalhousie University Animal Care Committee (24\u0026ndash;037). Mice were cared for in accordance with Canadian Council on Animal Care guidelines.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChemical synthesis\u003c/h2\u003e\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]BMP (\u003cb\u003e1\u003c/b\u003e), BMP (\u003cb\u003e2\u003c/b\u003e), 1-methylpiperidin-4-yl \u003cem\u003ep\u003c/em\u003e-nitrobenzoate (\u003cb\u003e3\u003c/b\u003e) and [\u003csup\u003e18\u003c/sup\u003eF]4-fluorobenzoic acid ([\u003csup\u003e18\u003c/sup\u003eF]FBA, \u003cb\u003e4\u003c/b\u003e) were synthesized (Supplementary Scheme 1) as described previously [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Details and analytical data are available in the Supplementary Material.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDynamic PET and MR Imaging\u003c/h3\u003e\n\u003cp\u003eDynamic PET imaging, following lateral tail vein injection of [\u003csup\u003e18\u003c/sup\u003eF]BMP, was completed in WT (three ♀ and four ♂), 5XFAD (three ♀ and three ♂) and BChE-KO (three ♀ and three ♂) mice with an average age of 13.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 months (Supplementary Table\u0026nbsp;1). PET imaging following injection of [\u003csup\u003e18\u003c/sup\u003eF]FBA was completed in a 13.6-month-old male, 5XFAD mouse. Details on mouse strain and PET/MR image methods are provided in the Supplementary Material.\u003c/p\u003e\n\u003ch3\u003eDynamic PET Data Analysis\u003c/h3\u003e\n\u003cp\u003eDynamic PET data were analyzed using PMOD (PMOD Technologies, Zurich, Switzerland, version 4.106) to evaluate and compare uptake and clearance of [\u003csup\u003e18\u003c/sup\u003eF]BMP and [\u003csup\u003e18\u003c/sup\u003eF]FBA in mice. Reconstructed PET DICOM files were opened in Amide (version 1.0.4) and exported as DIMCOM 3.0 via (X)MedCon files prior to being loaded into PMOD. For anatomical reference, PET and corresponding MRI data were fused using a rigid body transformation and visually inspected to ensure goodness of fit. Calibrated PET data (MBq/mm\u003csup\u003e3\u003c/sup\u003e) were converted into % injected dose per ml (%ID/ml) and %ID. Volumes of interest (VOIs) for the lungs, heart, liver and kidneys were generated using standardized ellipsoids placed within each organ. A standardized whole brain VOI for examining biodistribution was generated using the PMOD iso-contouring tool. All other analysis of brain data used the Ma-Benveniste-Mirrione template atlas [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Time-activity curve (TAC) data generated for each mouse was exported from PMOD and analyzed in Excel or Prism (Supplementary Material - Statistical Analysis section).\u003c/p\u003e\n\u003ch3\u003eMetabolite Analysis\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e analysis of [\u003csup\u003e18\u003c/sup\u003eF]BMP was performed 20 min after injection to assess the ligand and its metabolite(s) in the periphery and brain, as described previously (20). Details summarized in the Supplementary Material.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEx Vivo\u003c/b\u003e \u003cb\u003eAutoradiography\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eEx vivo\u003c/em\u003e autoradiography, following injection of [\u003csup\u003e18\u003c/sup\u003eF]BMP, was completed in WT (three ♀ and five ♂), 5XFAD (four ♀ and five ♂) and BChE-KO (one ♂) mice (Supplementary Table\u0026nbsp;2). A 20 min survival period was selected based on PET TACs. Details available in the Supplementary Material.\u003c/p\u003e\n\u003ch3\u003eButyrylcholinesterase Histochemistry\u003c/h3\u003e\n\u003cp\u003eBChE histochemistry was performed on brain tissue sections from all experimental mice as described previously [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Details available in the Supplementary Material.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eChemical Synthesis\u003c/h2\u003e\u003cp\u003eRadiosynthesis of [\u003csup\u003e18\u003c/sup\u003eF]BMP (\u003cb\u003e1\u003c/b\u003e) provided a decay corrected radiochemical yield of 30%, radiochemical purity of 97% (Supplementary Fig.\u0026nbsp;1) and a molar activity of 12.1 GBq/mmol at the end of synthesis, in line with a previous report [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The HPLC retention time of synthesized [\u003csup\u003e18\u003c/sup\u003eF]BMP was consistent with BMP (\u003cb\u003e2\u003c/b\u003e) (Supplementary Fig.\u0026nbsp;2), confirming the identity. BMP (\u003cb\u003e2\u003c/b\u003e) was synthesized in 86% yield with 98% purity (Supplementary Figs.\u0026nbsp;3\u0026ndash;11). 1-Methylpiperidin-4-yl \u003cem\u003ep\u003c/em\u003e-nitrobenzoate (\u003cb\u003e3\u003c/b\u003e) yielded 64% with 96% purity (Supplementary Figs.\u0026nbsp;12\u0026ndash;20). Spectroscopic data for compounds \u003cb\u003e2\u003c/b\u003e and \u003cb\u003e3\u003c/b\u003e (Supplementary Scheme 1) were consistent with their structure. [\u003csup\u003e18\u003c/sup\u003eF]FBA (\u003cb\u003e4\u003c/b\u003e) was synthesized by base hydrolysis of [\u003csup\u003e18\u003c/sup\u003eF]BMP, yielding 24% with 98% radiochemical purity (Supplementary Figs.\u0026nbsp;21\u0026ndash;22).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDynamic PET Data Analysis\u003c/h3\u003e\n\u003cp\u003eFollowing injection of [\u003csup\u003e18\u003c/sup\u003eF]BMP, its distribution was characterized in mice using dynamic PET imaging. Immediate time points demonstrated ligand perfusion of the inferior vena cava, heart and lungs followed by the brain, liver and, finally, kidneys. Robust brain uptake indicated [\u003csup\u003e18\u003c/sup\u003eF]BMP effectively crossed the blood-brain barrier (BBB). Brain, heart, lungs, liver and kidneys VOIs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) were used to generate biodistribution TACs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) from data standardized to percent of the injected dose per ml (%ID/ml). Heart and lung uptake was rapid, while that in the liver and kidneys was comparatively delayed. Washout of activity from the heart and lungs was rapid while clearance in the liver was slower. Uptake and washout in the kidneys varied between mice whereby in the majority, a gradual washout was observed over time while in other mice, uptake continued over the duration of the scan.\u003c/p\u003e\u003cp\u003eAveraged TAC data from PET scans illustrate the uptake and clearance of [\u003csup\u003e18\u003c/sup\u003eF]BMP in the brains of WT, 5XFAD and BChE-KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Distinct separation between the 5XFAD TAC and both WT and BChE-KO curves were apparent, whereby [\u003csup\u003e18\u003c/sup\u003eF]BMP clearance from the brains of female and male 5XFAD mice appeared to be slower than their WT counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These differences were typically apparent 10 min after injection of [\u003csup\u003e18\u003c/sup\u003eF]BMP and continued until 50 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The TACs for BChE-KO mice appeared comparable to WT mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eStatistical analysis of [\u003csup\u003e18\u003c/sup\u003eF]BMP dynamic PET data demonstrated several differences between strains and sex. Although the average C\u003csub\u003emax\u003c/sub\u003e did not differ between strains it was higher in female mice (1.5%ID) compared to male (1.2%ID, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Consequently, sexes were analyzed separately. The %ID in the brains of 5XFAD mice were significantly higher at timepoints throughout the PET scan (Supplementary Tables\u0026nbsp;3 and 4). Compared to WT counterparts, the %ID in the brains of female 5XFAD mice was 43.8% higher at 20 mins and 97.8% at 50 mins with significant differences at 10 and 20 mins post injection. In male 5XFAD mice, the brain %ID was increased 27.6% at 20 min and 67.5% at 50 mins compared to WT counterparts with significant differences at 10, 20, 30, 40 and 50 mins. The rates of clearance or half-life of ligand activity (t\u003csub\u003e1/2 clearance\u003c/sub\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were significantly longer in 5XFAD mice relative to WT counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Table\u0026nbsp;5). Compared to WT mice, the t\u003csub\u003e1/2 clearance\u003c/sub\u003e was 33% higher in female 5XFAD mice and 39% higher in males.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAverage data and standard deviation of the t\u003csub\u003e1/2 clearance\u003c/sub\u003e derived from an exponential decay curve model of 1-methyl-4-piperidinyl \u003cem\u003ep\u003c/em\u003e-fluorobenzoate ([\u003csup\u003e18\u003c/sup\u003eF]BMP) dynamic PET data in wild-type (WT), 5XFAD and butyrylcholinesterase knockout (BChE-KO) mice. The average R\u003csup\u003e2\u003c/sup\u003e with standard deviation is shown demonstrating the goodness of fit of the modelled curve.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003et\u003csub\u003e1/2 clearance\u003c/sub\u003e (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eFemale\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eWT\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e5XFAD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBChE-KO\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;\u0026lt;\u0026thinsp;.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eMale\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eWT\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e5XFAD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBChE-KO\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e4.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ePET imaging data in BChE-KO mice assessed off-target/non-specific interactions of [\u003csup\u003e18\u003c/sup\u003eF]BMP in the brain. Uptake, clearance (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and the %ID in the whole brain VOI (Supplementary Table\u0026nbsp;3) of male and female BChE-KO mice closely matched WT counterparts (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Statistical analysis showed no significant differences in the %ID in the brain at any timepoint (Supplementary Table\u0026nbsp;4) or t\u003csub\u003e1/2clearance\u003c/sub\u003e (Supplementary Table\u0026nbsp;5).\u003c/p\u003e\u003cp\u003ePET imaging of a male 5XFAD mouse injected with [\u003csup\u003e18\u003c/sup\u003eF]FBA, a radioactive [\u003csup\u003e18\u003c/sup\u003eF]BMP metabolite, showed limited BBB crossing and relatively quick washout from the brain (Supplementary Fig.\u0026nbsp;23). Compared to the average values of [\u003csup\u003e18\u003c/sup\u003eF]BMP in male 5XFAD mice, whole brain C\u003csub\u003emax\u003c/sub\u003e of [\u003csup\u003e18\u003c/sup\u003eF]FBA was 3.7x lower while the brain clearance (t\u003csub\u003e1/2 clearance\u003c/sub\u003e) was 21% faster. Peripheral biodistribution was similar for both ligands.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMetabolite Analysis\u003c/h2\u003e\u003cp\u003eBrain, liver and urine were analyzed for parent compound and metabolites 20 min after [\u003csup\u003e18\u003c/sup\u003eF]BMP injection in female WT, 5XFAD and BChE-KO mice. HPLC analysis of 5XFAD brain isolate showed clear presence of [\u003csup\u003e18\u003c/sup\u003eF]BMP, while WT and BChE-KO samples had lower activity and no clear [\u003csup\u003e18\u003c/sup\u003eF]BMP peaks. Liver isolate samples from all strains contained [\u003csup\u003e18\u003c/sup\u003eF]BMP and radioactive metabolites. Urine from all strains showed high activity from metabolites, with minor [\u003csup\u003e18\u003c/sup\u003eF]BMP detected. See Supplementary Fig.\u0026nbsp;24 for HPLC data.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003eautoradiography\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eEx vivo\u003c/em\u003e autoradiography with [\u003csup\u003e18\u003c/sup\u003eF]BMP showed clear differences between WT and 5XFAD mice. In WT brains, activity was mainly within white matter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) while the cortical grey matter, striatum, thalamus, hippocampus and brainstem showed less and remained uniform. Notably, brain regions with higher levels of BChE associated with neurons and neuropil [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] were not associated with increased retention. In contrast, 5XFAD mouse brain showed higher activity in the cortex, amygdala and the hippocampal formation, particularly the subiculum, regions with BChE-associated Aβ plaques (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Uptake in the 5XFAD hypothalamus, striatum, brainstem and cerebellum appeared generally consistent with WT counterparts. \u003cem\u003eEx vivo\u003c/em\u003e autoradiography in a 5.5-month-old 5XFAD female showed a similar pattern of retention compared to older mice (Supplementary Fig.\u0026nbsp;25). Additional data from male and female, WT and 5XFAD mice are provided in Fig.\u0026nbsp;25 of the Supplementary Material. \u003cem\u003eEx vivo\u003c/em\u003e autoradiography in the BChE-KO mouse appeared consistent with that in WT (Supplementary Fig.\u0026nbsp;25).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eButyrylcholinesterase Histochemistry\u003c/h2\u003e\u003cp\u003eThere was robust tissue staining of BChE enzymatic activity in the brain sections of 5XFAD and WT mice from PET imaging studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In WT, staining was associated with white matter, glia and select populations of neurons, as shown previously [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Staining of white matter, neuropil and neurons was also seen in 5XFAD brain sections but there was also staining associated with Aβ plaques (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), as shown previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. There was no staining of BChE activity in the brain sections from BChE-KO mice. Staining of brain tissue sections of mice from \u003cem\u003eex vivo\u003c/em\u003e autoradiography studies resulted in intense staining artefact associated with cell nuclei and poor staining of BChE activity associated with neurons, neuropil and Aβ plaques. Consequently, brain sections from mice used in PET imaging were compared to autoradiography shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current study evaluated 1-methyl-4-piperidinyl \u003cem\u003ep-\u003c/em\u003e[\u003csup\u003e18\u003c/sup\u003eF]fluorobenzoate ([\u003csup\u003e18\u003c/sup\u003eF]BMP) in WT, 5XFAD and BChE-KO mice for brain uptake and localization to BChE associated with Aβ plaques and normal neural elements (i.e. neurons, glia, neuropil, white matter) as well as off-target/non-specific interactions. [\u003csup\u003e18\u003c/sup\u003eF]BMP was synthesized efficiently and remained stable for PET imaging, metabolite and autoradiography studies. Despite peripheral metabolism, [\u003csup\u003e18\u003c/sup\u003eF]BMP crossed the BBB sufficiently for brain visualization (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This contrasted with its metabolite [\u003csup\u003e18\u003c/sup\u003eF]FBA, which showed poor BBB penetration and faster brain clearance (Supplementary Fig.\u0026nbsp;23). Brain clearance of [\u003csup\u003e18\u003c/sup\u003eF]BMP was significantly slower in 5XFAD mice compared to its WT counterpart, illustrated by the TACs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the %ID remaining in the brain at specific timepoints (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Tables\u0026nbsp;3 and 4) and t\u003csub\u003e1/2 clearance\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary Table\u0026nbsp;5). \u003cem\u003eIn vivo\u003c/em\u003e metabolite analysis indicated [\u003csup\u003e18\u003c/sup\u003eF]BMP was not metabolized in the brain (Supplementary Fig.\u0026nbsp;24). \u003cem\u003eEx vivo\u003c/em\u003e autoradiography confirmed 5XFAD brain retention of [\u003csup\u003e18\u003c/sup\u003eF]BMP in regions where BChE was associated with Aβ plaques but not normal neural elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Supplementary Fig.\u0026nbsp;25). These results indicate [\u003csup\u003e18\u003c/sup\u003eF]BMP specifically visualizes BChE-associated Aβ plaques and differentiates 5XFAD from WT mice.\u003c/p\u003e\u003cp\u003eCholinesterases (ChEs) associated with Aβ plaques have altered kinetic properties, including pH effects, inhibitor sensitivity and substrate affinities [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], that may affect an imaging agent\u0026rsquo;s ability to accurately reflect ChE distribution in the AD brain. For example, PET imaging with AChE-specific substrates such as [\u003csup\u003e11\u003c/sup\u003eC]MP4A and [\u003csup\u003e11\u003c/sup\u003eC]MP4P have successfully shown the distribution of this enzyme associated with neural structures in the normal and diseased brain [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] but did not recapitulate the distribution of AChE-associated Aβ plaques. Similarly, the BChE ligand [\u003csup\u003e11\u003c/sup\u003eC]MP4B showed generally decreased BChE levels in AD rather than the higher levels due to BChE-associated Aβ plaques [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, a highly potent BChE inhibitor ligand showed differences between 5XFAD and WT mice in younger age groups but not older, when BChE-associated Aβ plaques levels were higher [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These observations suggest ligand affinity for ChEs bound to normal neural elements and Aβ plaques may differ, possibly due to altered kinetic properties [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] resulting from conformational changes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consequently, an imaging agent that accurately recapitulates the levels of ChEs associated with normal neural elements may not interact with and successfully image ChEs associated with Aβ plaques and \u003cem\u003evice versa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eDynamic PET imaging and \u003cem\u003eex vivo\u003c/em\u003e autoradiography data suggests [\u003csup\u003e18\u003c/sup\u003eF]BMP may exhibit higher affinity for Aβ plaque bound BChE compared to that associated with normal neural elements. In WT mice, BChE is associated with glia, white matter and a distinct population of neurons [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Consequently, PET imaging of a BChE ligand would be expected to yield differing imaging profiles between WT and BChE-KO mice. However, our analysis of [\u003csup\u003e18\u003c/sup\u003eF]BMP between these two strains found no significant differences implying limited engagement of [\u003csup\u003e18\u003c/sup\u003eF]BMP with BChE-associated normal neural elements. These comparable results also suggest that brain retention in these strains may, at least partially, represent non-specific, off-target binding commonly seen with other brain imaging agents, including routinely used clinical amyloid ligands [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Such non-specific interactions are likely also present, to a degree, in the 5XFAD mouse. However, \u003cem\u003eex vivo\u003c/em\u003e autoradiography studies clearly demonstrate the increased retention of [\u003csup\u003e18\u003c/sup\u003eF]BMP observed in PET imaging studies is due to the localization of activity in regions exhibiting high levels of BChE-associated Aβ plaques, underlying the overall increased ligand retention in this mouse model of AD.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e enzyme kinetic behavior of [\u003csup\u003e18\u003c/sup\u003eF]BMP may account for its favorable imaging of BChE-associated with Aβ plaques. Our previous histochemical studies evaluating potential ChE imaging agents in brain tissue sections provided insights into their kinetic behavior when interacting with ChEs associated with normal neural elements and Aβ plaques [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These studies demonstrated that, although \u003cem\u003ein vitro\u003c/em\u003e kinetics showed BMP was a BChE-specific substrate with properties of a potential metabolic trapping imaging agent, it was not hydrolyzed well in the brain and may, in fact, engage BChE as an inhibitor [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The current work and previous reports [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], also indicates [\u003csup\u003e18\u003c/sup\u003eF]BMP is not hydrolyzed in the brain. These findings suggest [\u003csup\u003e18\u003c/sup\u003eF]BMP may have a prolonged interaction with BChE, acting as an inhibitor, and function as a molecular imaging agent capable of visualizing BChE associated with Aβ plaques. However, the mechanism of [\u003csup\u003e18\u003c/sup\u003eF]BMP interaction with BChE-associated Aβ plaques remains unclear. Previous studies have shown other compounds inhibit BChE-associated Aβ plaques by binding to remote allosteric sites of the enzyme [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. As [\u003csup\u003e18\u003c/sup\u003eF]BMP is not hydrolyzed in the brain, this ligand may engage BChE-associated Aβ plaques similarly.\u003c/p\u003e\u003cp\u003eThe bioavailability of [\u003csup\u003e18\u003c/sup\u003eF]BMP in WT, 5XFAD and BChE-KO mice appeared comparable. Peripheral metabolism of [\u003csup\u003e18\u003c/sup\u003eF]BMP in BChE-KO mice matched that in WT and 5XFAD mice suggesting that enzymes other than BChE may be largely responsible for its metabolism in the periphery. Comparable C\u003csub\u003emax\u003c/sub\u003e values indicate similar parent to metabolite ratio (i.e. [\u003csup\u003e18\u003c/sup\u003eF]BMP versus [\u003csup\u003e18\u003c/sup\u003eF]FBA) in the periphery. Dynamic PET imaging showed [\u003csup\u003e18\u003c/sup\u003eF]FBA, the main metabolite of [\u003csup\u003e18\u003c/sup\u003eF]BMP, does not sufficiently cross the BBB, resulting in significantly lower uptake and C\u003csub\u003emax\u003c/sub\u003e. Overall, [\u003csup\u003e18\u003c/sup\u003eF]BMP brain bioavailability appeared consistent across all strains of mice.\u003c/p\u003e\u003cp\u003eFuture studies will be required to validate [\u003csup\u003e18\u003c/sup\u003eF]BMP as a potential AD PET imaging ligand in humans. Regional analysis of brain uptake may clarify regions with intense \u003cem\u003ein vivo\u003c/em\u003e uptake (e.g. cerebral cortex) improving sensitivity and specificity for differentiating AD from non-AD brains rather than whole brain uptake in this study. \u003cem\u003eIn vivo\u003c/em\u003e metabolite analysis showed metabolism in the blood. Consequently, a metabolite correction will be necessary to derive PET kinetic parameters. Although the bioavailability of [\u003csup\u003e18\u003c/sup\u003eF]BMP was sufficient for brain imaging, determining the plasma free fraction may yield useful information as well. To show the value of [\u003csup\u003e18\u003c/sup\u003eF]BMP as an AD imaging agent, it will be necessary to test in mice at earlier age points than that used in this study. However, \u003cem\u003eex vivo\u003c/em\u003e autoradiography in a 5.5-month-old female 5XFAD mouse (Supplementary Fig.\u0026nbsp;25) showed retention comparable to older mice, suggesting potential AD pathology detection at earlier stages.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe current work provides a proof-of-principle that the [\u003csup\u003e18\u003c/sup\u003eF]BMP PET ligand can distinguish the 5XFAD amyloid mouse model from its WT counterpart by visualizing BChE-associated Aβ plaques. Molecular imaging of BChE-associated AD pathology may enhance the multi-modal approach of AD diagnosis, distinguishing cognitively normal individuals with plaques from those with AD, as well as present an avenue for monitoring of disease progression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eG. Andrew Reid: Contributed to completion of experiments, analysis and interpretation of data as well as drafting, revision and approving final content of manuscript.\u003c/p\u003e\n\u003cp\u003eDrew R. DeBay: Contributed to conception and design, analysis and interpretation of data as well as drafting, revision and approving final content of manuscript.\u003c/p\u003e\n\u003cp\u003eIan R. Macdonald: Contributed to the analysis and interpretation of the data as well as drafting, revision and approving the final content of the manuscript.\u003c/p\u003e\n\u003cp\u003eAntoun Bou Laouz: Contributed to ensuring availability of radioactive materials from the cyclotron, discussion about purity of radioactive raw material, reviewing information and data related to the radioactive materials, revising the manuscript and approving the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003eIan R. Pottie: Contributed to conception and design, funding acquisition, aquiring, analysis and interpretation of data and drafting, revision and approval of final content of manuscript.\u003c/p\u003e\n\u003cp\u003eSultan Darvesh: Project conceptualization, funding acquisition, supervision, data curation, validation and analysis, resources, writing, review, editing and approval of final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eWe would like to thank Dr. John Fisk and Meghan Cash for critically reading this manuscript and Hillary Maillet, Meagan Laffin, Emily MacAulay, Danielle Aucoin, Matt Sawler, Jessica George, Deanna Burns, K. Brewer, C. Davis for their technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This research was supported by the Canadian Institutes of Health Research (MOP-82798, RNS- 117795, MOP-119343, PJT – 153319), the Canadian Foundation for Innovation (Grant No. 37854), the Dalhousie Medical Research Foundation (DMRF Chemists, DMRF Clare Durland Fund in Alzheimer’s Disease Research; DMRF Research Grant – Robert and Barbara Pickett; DMRF Gillian’s Hope for MS Research Fund, Mrs. Sadie MacLeod through the Dalhousie Medical Research Foundation Adopt-a-Researcher program), and the Dalhousie University Endowed Irene MacDonald Sobey Endowed Chair in Curative Approaches to Alzheimer’s Disease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e: Sultan Darvesh is a scientific co-founder and stockholder in Treventis Corporation. \u0026nbsp; Sultan Darvesh, Ian R. Pottie and Ian R. Macdonald are listed as co-inventors on patents assigned to Treventis Corporation. \u0026nbsp;No other authors have financial disclosures or conflicts of interest with this submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e: All experiments involving animals were approved by the Dalhousie University Animal Care Committee (24-037). \u0026nbsp;Mice were cared for in accordance with Canadian Council on Animal Care guidelines. \u0026nbsp; This article does not contain any studies with human participants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e: \u0026nbsp;All research data are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFerrari C, Sorbi S (2021) The complexity of Alzheimer's disease: an evolving puzzle. 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J Nuc Med 66(Supp 2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/alz.14338\u003c/span\u003e\u003cspan address=\"10.1002/alz.14338\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDarvesh S, Cash MK, Forrestall K et al (2025) Differential senolytic inhibition of normal versus Aβ-associated cholinesterases: implications in aging and Alzheimer's disease. Aging 17(3):822\u0026ndash;850. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/aging.206227\u003c/span\u003e\u003cspan address=\"10.18632/aging.206227\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-imaging-and-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mibi","sideBox":"Learn more about [Molecular Imaging and Biology](http://link.springer.com/journal/11307)","snPcode":"11307","submissionUrl":"https://www.editorialmanager.com/mibi/default2.aspx","title":"Molecular Imaging and Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Alzheimer’s disease, PET imaging, ex vivo autoradiography, Aβ, biomarker, acetylcholinesterase","lastPublishedDoi":"10.21203/rs.3.rs-7491155/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7491155/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiagnosis of Alzheimer’s disease (AD) requires symptoms of dementia and accumulation of amyloid-β (Aβ) and tau in the brain. Molecular imaging of Aβ or tau in AD, though informative, is complicated by the finding that similar changes are found in brains of ~ 30% of cognitively normal older individuals. Butyrylcholinesterase (BChE), normally present in low levels in the cerebral cortex, is found in high levels associated with Aβ plaques in AD. When associated with this pathological structure, the biochemical properties of BChE are altered. The aim of the present study was to determine if [\u003csup\u003e18\u003c/sup\u003eF]1-Methyl-4-piperidinyl p-fluorobenzoate ([\u003csup\u003e18\u003c/sup\u003eF]BMP) can image BChE associated with Aβ in 5XFAD mouse model of AD and distinguish it from its wild-type (WT) counterpart.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProcedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]BMP was synthesized and evaluated in wild-type (WT), 5XFAD and BChE knock-out (BChE-KO) mouse models for \u003cem\u003ein vivo\u003c/em\u003e dynamic PET imaging of BChE. Time-activity curves were generated and [\u003csup\u003e18\u003c/sup\u003eF]BMP clearance parameters were determined. Brain, liver and urine homogenates were evaluated for [\u003csup\u003e18\u003c/sup\u003eF]BMP and its metabolites. \u003cem\u003eEx vivo\u003c/em\u003e autoradiography mapped the distribution of [\u003csup\u003e18\u003c/sup\u003eF]BMP brain retention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e PET imaging following injection of [\u003csup\u003e18\u003c/sup\u003eF]BMP demonstrated significantly greater brain retention of activity in the 5XFAD mouse model compared to WT, while BChE-KO mice mirrored WT levels. Metabolite analysis confirmed [\u003csup\u003e18\u003c/sup\u003eF]BMP was metabolized in the periphery but survived in sufficient quantity to enter the brain. \u003cem\u003eEx vivo\u003c/em\u003e autoradiography showed [\u003csup\u003e18\u003c/sup\u003eF]BMP retention in the 5XFAD mouse brain where BChE-associated Aβ plaques were prominent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese results demonstrate that PET imaging of BChE-associated Aβ plaques is feasible, offering an avenue to evaluate role(s) of BChE in AD pathogenesis, progression and complement the existing AD biomarker framework.\u003c/p\u003e","manuscriptTitle":"Molecular imaging of butyrylcholinesterase associated with amyloid-β plaques distinguishes 5XFAD from wild-type mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-23 11:45:16","doi":"10.21203/rs.3.rs-7491155/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-12-16T05:51:49+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-11-27T12:22:51+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-11T16:26:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-01T23:57:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Imaging and Biology","date":"2025-08-29T15:17:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-imaging-and-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mibi","sideBox":"Learn more about [Molecular Imaging and Biology](http://link.springer.com/journal/11307)","snPcode":"11307","submissionUrl":"https://www.editorialmanager.com/mibi/default2.aspx","title":"Molecular Imaging and Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"25760f10-7a24-4706-ada8-484ca6b34762","owner":[],"postedDate":"November 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T17:24:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-23 11:45:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7491155","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7491155","identity":"rs-7491155","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}