Increased monoamine oxidase-A (MAO-A) activity in Alzheimer’s disease (AD) may be detrimental to the point of neurodegeneration. To assess MAO-A activity in AD, we compared four biomarkers, Aβ plaques, tau, translocator protein (TSPO), and MAO-A in postmortem AD. Radiotracers were [18F]FAZIN3 for MAO-A, [18F]flotaza and [125I]IBETA for Aβ plaques, [124/125I]IPPI for tau, and [18F]FEPPA for TSPO imaging. Brain sections of the anterior cingulate (AC; gray matter GM) and corpus callosum (CC; white matter WM) from cognitively normal control (CN, n = 6) and AD (n = 6) subjects were imaged using autoradiography and immunostaining. Using competition with clorgyline and (R)-deprenyl, the binding of [18F]FAZIN3 was confirmed to be selective to MAO-A levels in the AD brain sections. Increases in MAO-A, Aβ plaque, tau, and TSPO activity were found in the AD brains compared to the control brains. The [18F]FAZIN3 ratio in AD GM versus CN GM was 2.80, suggesting a 180% increase in MAO-A activity. Using GM-to-WM ratios of AD versus CN, a >50% increase in MAO-A activity was observed (AD/CN = 1.58). Linear positive correlations of [18F]FAZIN3 with [18F]flotaza, [125I]IBETA, and [125I]IPPI were measured and suggested an increase in MAO-A activity with increases in Aβ plaques and tau activity. Our results support the finding that MAO-A activity is elevated in the anterior cingulate cortex in AD and thus may provide a new biomarker for AD in this brain region.
A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membrane-permeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in brain. Ligands, like varenicline, with high pK a and high affinity for α4β2-type nicotinic receptors (α4β2Rs) are trapped in intracellular acidic vesicles containing α4β2Rs in vitro . Nicotine, with lower pK a and α4β2R affinity, is not trapped. Here, we extend our results by imaging nicotinic PET ligands in vivo in male and female mouse brain and identifying the trapping brain organelle in vitro as Golgi satellites (GSats). Two PET 18 F-labeled imaging ligands were chosen: [ 18 F]2-FA85380 (2-FA) with varenicline-like pK a and affinity and [ 18 F]Nifene with nicotine-like pK a and affinity. [ 18 F]2-FA PET-imaging kinetics were very slow consistent with 2-FA trapping in α4β2R-containing GSats. In contrast, [ 18 F]Nifene kinetics were rapid, consistent with its binding to α4β2Rs but no trapping. Specific [ 18 F]2-FA and [ 18 F]Nifene signals were eliminated in β2 subunit knock-out (KO) mice or by acute nicotine (AN) injections demonstrating binding to sites on β2-containing receptors. Chloroquine (CQ), which dissipates GSat pH gradients, reduced [ 18 F]2-FA distributions while having little effect on [ 18 F]Nifene distributions in vivo consistent with only [ 18 F]2-FA trapping in GSats. These results are further supported by in vitro findings where dissipation of GSat pH gradients blocks 2-FA trapping in GSats without affecting Nifene. By combining in vitro and in vivo imaging, we mapped both the brain-wide and subcellular distributions of weak-base nicotinic receptor ligands. We conclude that ligands, such as varenicline, are trapped in neurons in α4β2R-containing GSats, which results in very slow release long after nicotine is gone after smoking. SIGNIFICANCE STATEMENT Mechanisms of nicotine addiction remain poorly understood. An earlier study using in vitro methods found that the anti-smoking nicotinic ligand, varenicline (Chantix) was trapped in α4β2R-containing acidic vesicles. Using a fluorescent-labeled high-affinity nicotinic ligand, this study provided evidence that these intracellular acidic vesicles were α4β2R-containing Golgi satellites (GSats). In vivo PET imaging with F-18-labeled nicotinic ligands provided additional evidence that differences in PET ligand trapping in acidic vesicles were the cause of differences in PET ligand kinetics and subcellular distributions. These findings combining in vitro and in vivo imaging revealed new mechanistic insights into the kinetics of weak base PET imaging ligands and the subcellular mechanisms underlying nicotine addiction.
A decline of norepinephrine transporter (NET) level is associated with several psychiatric and neurological disorders. Therefore positron emission tomography (PET) imaging agents are greatly desired to study the NET pathway. We have developed a C-fluoropropyl analog of nisoxetine: (R)-N-methyl-3-(3'-[(18)F]fluoropropyl)phenoxy)-3-phenylpropanamine ((18)F-MFP3) as a new potential PET radiotracer for NET with the advantage of the longer half-life of fluorine-18 (110 min compared with carbon-11 (20 min). Synthesis of (R)-N-methyl-3-(3'-fluoropropyl)phenoxy)-3-phenylpropanamine (MFP3) was achieved in five steps starting from (S)-N-methyl-3-ol-3-phenylpropanamine in approx. 3-5% overall yields. In vitro binding affinity of nisoxetine and MFP3 in rat brain homogenates labeled with (3)H-nisoxetine gave Ki values of 8.02 nM and 23 nM, respectively. For radiosynthesis of (18)F-MFP3, fluorine-18 was incorporated into a tosylate precursor, followed by the deprotection of the N-BOC-protected amine group with a 15% decay corrected yield in 2.5 h. Reverse-phase chromatographic purification provided (18)F-MFP3 in specific activities of >2000 Ci/mmol. Fluorine-18 labeled (18)F-MFP3 has been produced in modest radiochemical yields and in high specific activities. Evaluation of (18)F-MFP3 in animal imaging studies is in progress in order to validate this new fluorine-18 radiotracer for PET imaging of NET.
Transgenic mice line M83 that express the A53T mutant α–synuclein protein at six times the level of endogenous mice α–synuclein are a model of α-synucleinopathy found in Parkinson’s disease (PD). This Hualpha-Syn (A53T) PD model is useful in assessing non-motor deficits at earlier stages of onset of PD. We report findings on metabolic changes using [ 18 F]FDG PET/CT in the Hualpha-Syn (A53T) PD mouse model in comparison to non-carrier mice. Whole-body PET/CT imaging of male and female mice were carried out 2 h after [ 18 F]FDG ip administration under 3% isoflurane anesthesia. Brain images were analyzed with PET images coregistered to a mouse brain MRI template. Hualpha-Syn (A53T) mice had significantly lower [ 18 F]FDG uptake in several brain regions compared to the no-carrier mice. Significant hind limb muscle and lower spinal cord [ 18 F]FDG hypometabolism at 9 months of age in A53T PD mice was also indicative of neurodegenerative disease, with a progressive motoric dysfunction leading to death. Significant decrease (up to 30%) in [ 18 F]FDG uptake were observed in 9-month old male and female Hualpha-Syn (A53) mice. This is consistent with the cortical hypometabolism in PD patients. Hualpha-Syn (A53) mice may thus be a suitable model for studies related to PD α-synucleinopathy for the discovery of new biomarkers.
Nicotinic acetylcholine receptors (nAChRs) are involved in various central nervous system functions and have also been implicated in several neurodegenerative disorders. The heteromeric α4β2* and homomeric α7 are two major nAChR subtypes which have been studied in the brain using positron emission tomography (PET). Our comparative autoradiographic studies of the two receptor types in the mouse and rat brains show major differences in the thalamus (α4β2* >> α7), hippocampus (α7 >> α4β2*), and subiculum (α4β2* >> α7). A relatively newer heteromeric α7β2 nAChR subtype has been identified in the brain which may have a greater role in neurodegeneration. We report the development of KS7 (3-(2-(S)-azetidinylmethoxy)-5-(1,4-diaza-bicyclo[3.2.2]nonane)pyridine) which incorporates structural features of Nifzetidine (high affinity for α4β2* nAChR) and ASEM (high affinity for α7 nAChR) in an effort to target α7 and β2 subunits in α7β2 nAChR. KS7 exhibited higher affinities (IC50 = 50 to 172 nM) for [3H]cytisine radiolabeled sites and weaker affinities (IC50 = 10 μM) for [125I]-α-bungarotoxin radiolabeled rat brain sites in several brain regions. The weaker affinity of KS7 to α7 nAChR may suggest lack of binding at the α7 subunit of α7β2 nAChR. A radiolabeled derivative of KS7 may be required to identify any specific binding to brain regions suggested to contain α7β2 nAChR.
411 Objectives Histone deacetylase has been imaged using 18F-FAHA and may serve as a marker to study epigenetics. We report evaluation of 18F-FAHA as a probe in the early diagnosis of lung cancer using longitudinal MicroPET/MicroCT 18F-FAHA studies of A/J mice treated with NNK. Methods 18F-FAHA radiosynthesis was carried out in specific activity of ~2 Ci/μmol as reported (Mukhopadhyay 2006). A/J mice acquired from Jackson Labs were divided into 2 groups: 1. Controls for CT/PET; 2. NNK treatment group for CT/PET. Group 2 animals received NNK (100 mg/kg, ip, weekly for 4 wks). Mice were injected 50-100 μCi i.v. 18F-FAHA and then scanned in Inveon MicroPET/CT under anesthesia using 2.0% isoflurane. Ex vivo PET/CT scans of lungs were also obtained on some mice after the in vivo scans. Sections (5 to 10μm thick) of the isolated lungs were prepared for H&E staining and autoradiographic studies. Results MicroCT revealed presence of lung nodules in 8 mo old mice while control mice were free of tumors. Little uptake of 18F-FAHA was in the control mice lungs while significant amount of 18F-FAHA was seen in the lungs of NNK treated mice. Quantitative analysis of the extent and amount of 18F-FAHA binding provided in vivo lung tumor/nontumor = >2.0. Studies of control A/J mice lungs showed little binding of 18F-FAHA, uniformly distributed. Ex vivo scans of the excised NNK and control mice lungs confirmed presence of extensive amounts of lung nodules seen by CT and confirmed by 18F-FAHA in the NNK mice while no tumors were detected in the control mice. Conclusions Our preliminary imaging studies with A/J mice lung cancer model suggests 18F-FAHA PET may allow the study of epigenetic mechanisms involved in NNK induced tumorigenesis in the lungs. Further studies on the ability of 18F-FAHA to detect the lung tumors earlier are underway
1891 Objectives The acetylcholinesterase inhibitors (AChEIs) are used in management of Alzheimer9s disease in humans. Previous in vitro studies found a decrease in binding of α4β2 receptor agonist [18F]nifene in the presence of combined ACh and AChEIs but no effects of AChEIs alone (Easwaramoorthy et al, 2007). Our goal for this study was to evaluate the effects of AChEIs on in vivo binding of 18F]nifene in the rat brain using PET/CT imaging. Methods Two male Sprague-Dawley rats (148 ± 2 g) were injected iv with [18F]nifene (13.8 ± 4.3 MBq) and received 90 min dynamic PET scans on two different days. The first day scan was a baseline scan; the second day scan was preceded by iv administration of Donepezil (1.0 mg/Kg), a potent AChEI, at 15 min before [18F]nifene. All PET images were co-registered to a rat MR template spatially normalized to Paxinos-Watson space. Volumes of interest (VOIs) from regions with high density of α4β2 receptors were drawn on the template and transferred to the PET images. Three VOIs were considered: thalamus (TH), frontal cortex (FC), and subiculum (SUB). Graphical analysis was used to compute the binding potentials (BPND) of [18F]nifene. Cerebellum was used as a reference region. Results The baseline BPNDs of [18F]nifene were 1.51 ± 0.05 (TH), 1.09 ± 0.04 (FC), and 0.76 ± 0.06 (SUB). The drug condition BPNDs were 1.79 ± 0.21 (TH), 1.16 ± 0.01 (FC), and 0.83 ± 0.04 (SUB). As a result the average increases in BPND in the presence of Donepezil were (18.33 ± 17.94) % (TH), 6.10 ± 4.31 % (FC), and 9.28 ± 3.72 % (SUB). Conclusions Donepezil at 1.0 mg/kg did not cause a decrease in binding of [18F]nifene in response to an expected increase in ACh levels. Effects of dose and timing of AChEI administration before [18F]nifene, as well as those of anesthesia are being considered as variables. Other AChEIs are also being tested. Research Support NIH AG02947
