Marie Svedberg

Selective Nicotinic Receptor Consequences in APPSWE Transgenic Mice

The nicotinic (nAChRs) and muscarinic (mAChRs) acetylcholine receptors and acetylcholinesterase (AChE) activity were studied in the brains of APP(SWE) transgenic mice (Tg+) and age-matched nontransgenic controls (Tg-) that were between 4 and 19 months of age. A significant increase in the binding of 125I-labeled alpha-bungarotoxin (alpha7 nAChRs) was observed in most brain regions analyzed in 4-month-old Tg+ mice, preceding learning and memory impairments and amyloid-beta (Abeta) pathology. The enhanced alpha7 receptor binding was still detectable at 17-19 months of age. Increase in [3H]cytisine binding (alpha4beta2 nAChRs) was measured at 17-19 months of age in Tg+ mice, at the same age when the animals showed heavy Abeta pathology. No significant changes in [3H]pirenzepine (M1 mAChRs) or [3H]AFDX 384 (M2 mAChRs) binding sites were found at any age studied. The upregulation of the nAChRs probably reflects compensatory mechanisms in response to Abeta burden in the brains of Tg+ mice.

Beta-amyloid causes downregulation of calcineurin in neurons through induction of oxidative stress

Calcineurin is an abundant cytosolic protein that is implicated in the modulation of glutamate release. Here we show that the expression level of this enzyme is reduced in primary neuronal cultures treated with beta-amyloid. Parallel experiments in ETNA cell lines expressing SOD1 suggested that the effect of beta-amyloid on calcineurin expression is mediated by oxidative stress. The relevance of the in vitro experiments was assessed by analysis of tissue from patients with Alzheimers disease (AD) and tissue from two strains of transgenic mice that mimic aspects of AD. The tissue from the AD brains displayed a pronounced downregulation of calcineurin immunoreactivity in profiles that were negative for glial fibrillary acidic protein (GFAP). In the hippocampus of the transgenic animals (which were analyzed in an early stage of the disease) the downregulation of calcineurin was restricted to mossy fiber terminals. A downregulation of the presynaptic pool of calcineurin may contribute to the dysregulation of glutamate release that is considered a hallmark of AD.

Upregulation of neuronal nicotinic receptor subunits α4, β2, and α7 in transgenic mice overexpressing human acetylcholinesterase

Neuronal nicotinic receptor binding sites as well as mRNA levels encoding for subunits α4, β2, and α7 were analysed in 3-mo-old transgenic mice generated with a neuronal overexpression of human acetylcholinesterase and in age-matched controls. The acetylcholinesterase transgenic mice display progressive cognitive impairment in spatial learning and memory. We here report a significantly increased [3H]epibatidine and [125I]αbungarotoxin binding in the cortex and the caudate putamen of these mice. Quantitative in situ hybridization showed significant upregulation of mRNA corresponding to the nicotinic receptor subunits α4, β2, and α7 in various brain regions in the transgenic mice compared to nontransgenic controls. Our results suggest that disruption of balanced cholinergic transmission by constitutive overexpression of acetylcholinesterase is accompanied by variable upregulation of several nicotinic receptor subtypes, in particular these associated with cholinergic terminals participating in compensatory response.

Characterization of [11C]Cimbi-36 as an agonist PET radioligand for the 5-HT2A and 5-HT2C receptors in the nonhuman primate brain

Only recently the first successful serotonin 2A (5-HT2A) receptor agonist PET radioligands have been described, with [(11)C]Cimbi-36 reported as the most promising in the pig brain so far. Agonist radioligands may target specifically the G protein-coupled state of the receptors and thereby provide a more meaningful assessment of available receptors than antagonist radioligands. In the current study we characterized [(11)C]Cimbi-36 receptor binding in the primate brain. On five experimental days, a total of 14 PET measurements were conducted in three female rhesus monkeys. On each day, PET measurements were conducted after intravenous injection of [(11)C]Cimbi-36 during baseline conditions and after intravenous infusion of the 5-HT2 receptor antagonist ketanserin (n=3) or the 5-HT2C receptor antagonist SB 242084 (n=2). On four of the experimental days an additional baseline PET measurement was conducted after injection of [(11)C]MDL 100907. All PET measurements were performed for 2h in a HRRT PET system and arterial blood was obtained for measurement of the [(11)C]Cimbi-36 input function. Quantification of [(11)C]Cimbi-36 receptor binding was performed using kinetic and graphical analysis. After injection of [(11)C]Cimbi-36 the regional distribution of radioactivity in brain was in accordance with the known 5-HT2 receptor distribution. The two-tissue compartment model was superior for the description of the time-radioactivity curves of all examined brain regions. BPND values obtained with reference tissue models correlated with corresponding values obtained with kinetic modeling. Administration of ketanserin decreased the binding in all brain regions but did not affect the cerebellar distribution volume. The BPND of [(11)C]Cimbi-36 was 56±8% of [(11)C]MDL 100907 across cortical regions, but higher in other brain regions including choroid plexus. After administration of SB 242084, [(11)C]Cimbi-36 binding was nearly completely inhibited in the choroid plexus, partly reduced in several subcortical regions (e.g. hippocampus), but not affected in the cortical regions. In conclusion, the receptor binding of [(11)C]Cimbi-36 can be quantified using kinetic modeling and the cerebellum was found to be a suitable reference region. The difference between [(11)C]Cimbi-36 and [(11)C]MDL 100907 binding in the choroid plexus is related to 5-HT2C receptor binding of [(11)C]Cimbi-36. [(11)C]Cimbi-36 is the first agonist radioligand suitable for examination of 5-HT2A receptors in the cortical regions and of 5-HT2C receptors in the choroid plexus of the primate brain.

Neuronal nicotinic and muscarinic receptor subtypes at different ages of transgenic mice overexpressing human acetylcholinesterase

Subtypes of nicotinic (alpha4 and alpha7) as well as muscarinic (M1 and M2) receptor binding sites were quantified in the brain of transgenic mice overexpressing human acetylcholinesterase (AChE) at different ages using selective radioligands. A significant increase in [(3)H]cytisine (alpha4) binding was found in the cortex and striatum of AChE transgenic (hAChE-Tg) mice from 3 days to 12 months of age in comparison to non-transgenic mice. In addition a significant increase in [(3)H]AF-DX-384 (M2) binding was found in the striatum of hAChE-Tg mice at 3 months of age compared to controls. No major alteration was observed in the [(125)I]alpha-bungarotoxin (alpha7) or the [(3)H]pirenzepine (M1) binding sites. The persistent increase in alpha4 and M2 receptor binding sites in hAChE-Tg mice suggests that these receptor subtypes may play an important role in compensatory mechanisms facilitating the impaired cholinergic neurotransmission in hAChE-Tg.

Effect of subchronic galantamine treatment on neuronal nicotinic and muscarinic receptor subtypes in transgenic mice overexpressing human acetylcholinesterase

Overexpression of acetylcholinesterase (AChE) in mice causes cholinergic deficits with memory impairment. In this study, AChE overexpressing (hAChE-Tg) and control (FVB/N) mice were treated with the AChE inhibitor (AChEI) galantamine (4 mg/kg/day) for 10 days. The concentration of galantamine in plasma was 75-80 ng/ml. The inhibition of AChE was 20% in red blood cells (RBC) and 30% in brain cortical tissue. A significant increase in [(3)H]cytisine (alpha4 nicotinic receptor) binding was measured in the CA1 and CA3 area of the hippocampus of FVB/N mice following galantamine treatment. Similarly, a significant increase in [(125)I]alphabungarotoxin (alpha7 nicotinic receptor) binding was found in the frontal cortex, retrosplenial gr. cortex, motor cortex and thalamus in galantamine treated FVB/N compared to saline treated mice. No significant changes in nicotinic receptor binding sites were observed in galantamine treated hAChE-Tg mice. Significant decreases in the muscarinic receptors measured by [(3)H]AF-DX-384 (M2 muscarinic receptor) and [(3)H]pirenzepine (M1 muscarinic receptor) were observed in several brain regions of galantamine treated FVB/N and hAChE-Tg mice. This study shows regional and receptor subtype specific changes in the nicotinic receptor subtypes compared to the muscarinic receptors following galantamine treatment in FVB/N and hAChE-Tg mice.

Comparative binding properties of the tau PET tracers THK5117, THK5351, PBB3, and T807 in postmortem Alzheimer brains

BackgroundThe aim of this study was to compare the binding properties of several tau positron emission tomography tracers—THK5117, THK5351, T807 (also known as AV1451; flortaucipir), and PBB3—head to head in the same human brain tissue.MethodsBinding assays were performed to compare the regional distribution of 3H-THK5117 and 3H-THK5351 in postmortem tissue from three Alzheimer’s disease (AD) cases and three control subjects in frontal and temporal cortices as well as in the hippocampus. Competition binding assays between THK5351, THK5117, PBB3, and T807, as well as off-target binding of THK5117 and T807 toward monoamine oxidase B (MAO-B), were performed using binding assays in brain homogenates and autoradiography of three AD cases.ResultsRegional binding of 3H-THK5117 and 3H-THK5351 was similar, except in the temporal cortex, which showed higher 3H-THK5117 binding. Saturation studies demonstrated two binding sites for 3H-THK5351 (Kd1 = 5.6 nM, Bmax = 76 pmol/g; Kd2 = 1 nM, Bmax = 40 pmol/g). Competition studies in the hippocampus between 3H-THK5351 and unlabeled THK5351, THK5117, and T807 revealed super-high-affinity sites for all three tracers (THK5351 Ki = 0.1 pM; THK5117 Ki = 0.3 pM; T807 Ki = 0.2 pM) and an additional high-affinity site (THK5351 Ki = 16 nM; THK5117 Ki = 20 nM; T807 Ki = 78nM). 18F-T807, 11C-THK5351, and 11C-PBB3 autoradiography of large frozen sections from three AD brains showed similar regional binding for the three tracers, with lower binding intensity for 11C-PBB3. Unlabeled THK5351 and T807 displaced 11C-THK5351 to a similar extent and a lower extent, respectively, compared with 11C-PBB3. Competition with the MAO-B inhibitor 3H-l-deprenyl was observed for THK5117 and T807 in the hippocampus (THK5117 Ki = 286 nM; T807 Ki = 227 nM) and the putamen (THK5117 Ki = 148 nM; T807 Ki = 135 nM). 3H-THK5351 binding was displaced using autoradiography competition with unlabeled THK5351 and T807 in cortical areas by 70–80% and 60–77%, respectively, in the basal ganglia, whereas unlabeled deprenyl displaced 3H-THK5351 binding by 40% in the frontal cortex and 50% in the basal ganglia.ConclusionsTHK5351, THK5117, and T807 seem to target similar binding sites, but with different affinities, whereas PBB3 seems to target its own binding site. Both THK5117 and T807 demonstrated off-target binding in the hippocampus and putamen with a ten times lower binding affinity to the MAO-B inhibitor deprenyl compared with 3H-THK5351.

In Vivo and In Vitro Characterization of a Novel MAO-B Inhibitor Radioligand, 18F-Labeled Deuterated Fluorodeprenyl

The aim of this study was to radiolabel a novel bis-deuterium substituted l-deprenyl analog (fluorodeprenyl-D2) with 18F and to evaluate its potential to visualize and quantify monoamine oxidase (MAO) B activity in vivo. Methods: The precursor compound (5a + 5b) and reference standard (6) were synthesized in multistep syntheses. Recombinant human MAO-B and MAO-A enzyme preparations were used to determine inhibitory concentrations of 50%. Radiolabeling was accomplished by a nucleophilic substitution reaction. Whole-hemisphere autoradiography was performed with 18F-fluorodeprenyl-D2. A PET study was performed on a cynomolgus monkey. Radiometabolites were measured in monkey plasma using high-performance liquid chromatography. Results: The 50% inhibitory concentration of compound 6 for MAO-B was 227 ± 36.8 nM. Radiolabeling was accomplished with high radiochemical yield, purity, and specific radioactivity. The autoradiography binding density of 18F-fluorodeprenyl-D2 was consistent with known MAO-B expression in the human brain. In vivo, 18F-fluorodeprenyl-D2 showed favorable kinetic properties, with relatively fast washout from the brain. Regional time–activity curves were better described by the 2-tissue-compartment model. Administration of a 1 mg/kg dose of l-deprenyl yielded 70% inhibition of MAO-B in all regions. Radiometabolite studies demonstrated 20% unchanged radioligand at 120 min after injection. 18F-fluorodeprenyl-D2 showed less irreversibility than did previously reported MAO-B radioligands. Conclusion: The results suggest that 18F-fluorodeprenyl-D2 is a suitable PET radioligand for visualization of MAO-B activity in the human brain.

Development of [11C]/[3H]THK-5351 – A potential novel carbon-11 tau imaging PET radioligand

INTRODUCTION Due to the rise in the number of patients with dementia the imperative for finding new diagnostic and treatment options becomes ever more pressing. While significant progress has been made in PET imaging of Aβ aggregates both in vitro and in vivo, options for imaging tau protein aggregates selectively are still limited. Based on the work previously published by researchers from the Tohoku University, Japan, that resulted in the development of [18F]THK-5351, we have undertaken an effort to develop a carbon-11 version of the identical structure - [11C]THK-5351. In parallel, THK-5351 was also labeled with tritium ([3H]THK-5351) for use in in vitro autoradiography (ARG). METHODS The carbon-11 labeling was performed starting with di-protected enantiomeric pure precursor - tert-butyl 5-(6-((2S)-3-fluoro-2-(tetrahydro-2H-pyran-2-yloxy)propoxy)quinolin-2-yl)pyridin-2-yl carbamate, which was reacted with [11C]MeI, using DMF as the solvent and NaH as base, followed by deprotection with trifluoroacetic acid/water mixture, resulting in enantiomerically pure carbon-11 radioligand, [11C]THK-5351 - (S)-1-fluoro-3-(2-(6-([11C]methylamino)pyridin-3-yl)quinolin-6-yloxy)propan-2-ol. Tritium labeling and purification of [3H]THK-5351 were undertaken using similar approach, resulting in [3H]THK-5351 with RCP >99.8% and specific radioactivity of 1.3GBq/μmol. RESULTS [11C]THK-5351 was produced in good yield (1900±355MBq), specific radioactivity (SRA) (361±119GBq/μmol at EOS+20min) and radiochemical purity (RCP) (>99.8%), with enantiomeric purity of 98.7%. [3H]THK-5351 was evaluated for ARG of tau binding in post-mortem human brain tissue using cortical sections from one AD patient and one control subject. [3H]THK-5351 binding density was higher in the AD patient compared to the control subject, the binding was displaced by unlabeled THK-5351 confirming specific [3H]THK-5351 binding.

Amyloid binding properties of curcumin analogues in Alzheimer’s disease postmortem brain tissue

The presence of β-amyloid (Aβ) containing plaques in the brain is a hallmark of Alzheimers disease (AD) and serves as a biomarker for confirmation of diagnosis postmortem. Early diagnosis is of great importance for optimal treatment and for monitoring disease progression in the brain. Highly specific and sensitive biomarkers are thus greatly needed to assess therapeutic efficacy, not only clinically, but also in terms of clearance of histopathological lesions and decelerated neurodegeneration. The objective of the present study was to give more insight into the binding of curcumin analogues, curcuminoids, to Aβ containing plaques in postmortem tissue from AD patients. In vitro autoradiography was utilized to explore affinity and displacement of the curcuminoids; curcumin, demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC) and dimethoxycurcumin (DIMC). We found that BDMC had the highest affinity for Aβ containing plaques in cortical AD brain tissue in comparison to other curcuminoids. Subsequently, [(3)H]BDMC showed significantly higher specific binding in cortical AD brain tissue compared to control subjects. These findings suggest that curcumin analogues, especially BDMC, may serve as a potential radioligands for Aβ plaque neuroimaging.