Athanasios Metaxas

An integrated proteomics approach shows synaptic plasticity changes in an APP/PS1 Alzheimer's mouse model

The aim of this study was to elucidate the molecular signature of Alzheimers disease-associated amyloid pathology. We used the double APPswe/PS1ΔE9 mouse, a widely used model of cerebral amyloidosis, to compare changes in proteome, including global phosphorylation and sialylated N-linked glycosylation patterns, pathway-focused transcriptome and neurological disease-associated miRNAome with age-matched controls in neocortex, hippocampus, olfactory bulb and brainstem. We report that signalling pathways related to synaptic functions associated with dendritic spine morphology, neurite outgrowth, long-term potentiation, CREB signalling and cytoskeletal dynamics were altered in 12 month old APPswe/PS1ΔE9 mice, particularly in the neocortex and olfactory bulb. This was associated with cerebral amyloidosis as well as formation of argyrophilic tangle-like structures and microglial clustering in all brain regions, except for brainstem. These responses may be epigenetically modulated by the interaction with a number of miRNAs regulating spine restructuring, Aβ expression and neuroinflammation. We suggest that these changes could be associated with development of cognitive dysfunction in early disease states in patients with Alzheimers disease.

Synthesis, structure activity relationship, radiolabeling and preclinical evaluation of high affinity ligands for the ion channel of the N-methyl-d-aspartate receptor as potential imaging probes for positron emission tomography.

The N-methyl-d-aspartate receptor (NMDAr) is involved in many neurological and psychiatric disorders including Alzheimers disease and schizophrenia. Currently, it is not possible to assess NMDAr availability in vivo. The purpose of this study was to develop a positron emission tomography (PET) ligand for the NMDAr ion channel. A series of di- and tri-N-substituted diarylguanidines was synthesized. In addition, in vitro binding affinity for the NMDAr ion channel in rat forebrain membrane fractions was assessed. Compounds 10, 11 and 32 were radiolabeled with either carbon-11 or fluorine-18. Ligands [(11)C]10 and [(18)F]32 were evaluated ex vivo in B6C3 mice. Biodistribution studies showed higher uptake of [(11)C]10 and [(18)F]32 in forebrain regions compared with cerebellum. In addition, for [(11)C]10 54% and for [(18)F]32 70% of activity in the brain at 60min was due to intact tracer. Pre-treatment with MK-801 (0.6mg·kg(-1), ip) slightly decreased uptake in NMDAr-specific regions for [(18)F]32, but not for [(11)C]10. As such [(18)F]32 has the best characteristics as a PET tracer for the ion channel of the NMDAr.

Quantification of the novel N-methyl-d-aspartate receptor ligand [11C]GMOM in man

[11C]GMOM (carbon-11 labeled N-(2-chloro-5-thiomethylphenyl)-N′-(3-[11C]methoxy-phenyl)-N′-methylguanidine) is a PET ligand that binds to the N-methyl-d-aspartate receptor with high specificity and affinity. The purpose of this first in human study was to evaluate kinetics of [11C]GMOM in the healthy human brain and to identify the optimal pharmacokinetic model for quantifying these kinetics, both before and after a pharmacological dose of S-ketamine. Dynamic 90 min [11C]GMOM PET scans were obtained from 10 subjects. In six of the 10 subjects, a second PET scan was performed following an S-ketamine challenge. Metabolite corrected plasma input functions were obtained for all scans. Regional time activity curves were fitted to various single- and two-tissue compartment models. Best fits were obtained using a two-tissue irreversible model with blood volume parameter. The highest net influx rate (Ki) of [11C]GMOM was observed in regions with high N-methyl-d-aspartate receptor density, such as hippocampus and thalamus. A significant reduction in the Ki was observed for the entire brain after administration of ketamine, suggesting specific binding to the N-methyl-d-aspartate receptors. This initial study suggests that the [11C]GMOM could be used for quantification of N-methyl-d-aspartate receptors.

Preclinical evaluation of [18F]PK-209, a new PET ligand for imaging the ion-channel site of NMDA receptors

INTRODUCTION The present study was designed to assess whether [(18)F]PK-209 (3-(2-chloro-5-(methylthio)phenyl)-1-(3-([(18)F]fluoromethoxy)phenyl)-1-methylguanidine) is a suitable ligand for imaging the ion-channel site of N-methyl-D-aspartate receptors (NMDArs) using positron emission tomography (PET). METHODS Dynamic PET scans were acquired from male rhesus monkeys over 120min, at baseline and after the acute administration of dizocilpine (MK-801, 0.3mg/kg; n=3/condition). Continuous and discrete arterial blood samples were manually obtained, to generate metabolite-corrected input functions. Parametric volume-of-distribution (VT) images were obtained using Logan analysis. The selectivity profile of PK-209 was assessed in vitro, on a broad screen of 79 targets. RESULTS PK-209 was at least 50-fold more selective for NMDArs over all other targets examined. At baseline, prolonged retention of radioactivity was observed in NMDAr-rich cortical regions relative to the cerebellum. Pretreatment with MK-801 reduced the VT of [(18)F]PK-209 compared with baseline in two of three subjects. The rate of radioligand metabolism was high, both at baseline and after MK-801 administration. CONCLUSIONS PK-209 targets the intrachannel site with high selectivity. Imaging of the NMDAr is feasible with [(18)F]PK-209, despite its fast metabolism. Further in vivo evaluation in humans is warranted.

Effect of aging and Alzheimer's disease-like pathology on brain monoamines in mice

&NA; Aging is the greatest single risk factor of the neurodegenerative disorder Alzheimers disease (AD). The monoaminergic system, including serotonin (5‐HT), dopamine (DA) and noradrenaline (NA) modulates cognition, which is affected in AD. Changes in monoamine levels have been observed in AD, but these can both be age‐ and/or disease‐related. We examined whether brain monoamine levels change as part of physiological aging and/or AD‐like disease in APPSWE/PS1&Dgr;E9 (APP/PS1) transgenic mice. The neocortex, hippocampus, striatum, brainstem and cerebellum of 6‐, 12‐, 18‐ and 24‐month‐old B6C3 wild‐type (WT) mice and of 18‐month old APP/PS1 and WT mice were analysed for 5‐HT, DA and NA contents by high pressure liquid chromatography (HPLC), along with neocortex from 14‐month‐old APP/PS1 and WT mice. While, we observed no aging effect in WT mice, we detected region‐specific changes in the levels of all monoamines in 18‐month‐old transgenic compared with WT mice. This included reductions in 5‐HT (−30%), DA (−47%) and NA (−32%) levels in the neocortex and increases of 5‐HT in the brainstem (+18%). No changes were observed in any of the monoamines in the neocortex from 14‐month‐old APP/PS1 mice. In combination, these findings indicate that aging alone is not sufficient to affect brain monoamine levels, unlike the APPSWE/PS1&Dgr;E9 genotype. HighlightsPhysiological aging is not associated with changes in the levels of brain monoamines.The APP/PS1 genotype reduces cortical levels of monoamines with increased aging.The APP/PS1 induced reduction in the cortex is paralleled by increases in subcortical monoamine levels.Aging does not necessarily precede changes in brain monoamines observed in AD‐like disease.

TNFα affects CREB-mediated neuroprotective signaling pathways of synaptic plasticity in neurons as revealed by proteomics and phospho-proteomics

Neuroinflammation is a hallmark of Alzheimers disease and TNFα as the main inducer of neuroinflammation has neurodegenerative but also pro-regenerative properties, however, the dose-dependent molecular changes on signaling pathway level are not fully understood. We performed quantitative proteomics and phospho-proteomics to target this point. In HT22 cells, we found that TNFα reduced mitochondrial signaling and inhibited mTOR protein translation signaling but also led to induction of neuroprotective MAPK-CREB signaling. Stimulation of human neurons with TNFα revealed similar cellular mechanisms. Moreover, a number of synaptic plasticity-associated genes were altered in their expression profile including CREB. SiRNA-mediated knockdown of CREB in human neurons prior to TNFα stimulation led to a reduced number of protein/phospho-protein hits compared to siRNA-mediated knockdown of CREB or TNFα stimulation alone and countermeasured the reduced CREB signaling. In vivo data of TNFα knockout mice showed that learning ability did not depend on TNFα per se but that TNFα was essential for preserving the learning ability after episodes of lipopolysaccharide-induced neuroinflammation. This may be based on modulation of CREB/CREB signaling as revealed by the in vitro / in vivo data. Our data show that several molecular targets and signaling pathways induced by TNFα in neurons resemble those seen in Alzheimers disease pathology.Neuroinflammation is a hallmark of Alzheimers disease and TNFα as the main inducer of neuroinflammation has neurodegenerative but also pro-regenerative properties, however, the dose-dependent molecular changes on signaling pathway level are not fully understood. We performed quantitative proteomics and phospho-proteomics to target this point.In HT22 cells, we found that TNFα reduced mitochondrial signaling and inhibited mTOR protein translation signaling but also led to induction of neuroprotective MAPK-CREB signaling. Stimulation of human neurons with TNFα revealed similar cellular mechanisms. Moreover, a number of synaptic plasticity-associated genes were altered in their expression profile including CREB.SiRNA-mediated knockdown of CREB in human neurons prior to TNFα stimulation led to a reduced number of protein/phospho-protein hits compared to siRNA-mediated knockdown of CREB or TNFα stimulation alone and countermeasured the reduced CREB signaling. In vivo data of TNFα knockout mice showed that learning ability did not depend on TNFα per se but that TNFα was essential for preserving the learning ability after episodes of lipopolysaccharide-induced neuroinflammation. This may be based on modulation of CREB/CREB signaling as revealed by the in vitro / in vivo data.Our data show that several molecular targets and signaling pathways induced by TNFα in neurons resemble those seen in Alzheimer´s disease pathology.

Synthesis and preclinical evaluation of carbon-11 labelled N-((5-(4-fluoro-2-[11C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine as a PET tracer for NR2B subunit-containing NMDA receptors

INTRODUCTION The N-methyl-D-Aspartate (NMDA) receptor plays an important role in learning and memory. Overactivation is thought to play an important role in neurodegenerative disorders such as Alzheimers disease. Currently, it is not possible to assess N-methyl-D-aspartate receptor (NMDAr) bio-availability in vivo. The purpose of this study was to develop a positron emission tomography (PET) ligand for the NR2B binding site of the NMDA receptor. METHODS N-((5-(4-fluoro-2-methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was radiolabelled with carbon-11 in the phenyl moiety. Biodistribution and blocking studies were carried out in anaesthetized mice and in non-anaesthetized rats. RESULTS N-((5-(4-fluoro-2-[(11)C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was prepared in 49±3% (decay-corrected) yield, affording 4.1±0.3 GBq of formulated product at the end of synthesis with a radiochemical purity of >99% and with a specific activity of 78±10 GBq/μmol. CONCLUSION A new NR2B PET ligand was developed in high yield. [(11)C]4 readily enters the brain and binds to the NR2B subunit-containing NMDAr in the rodent brain. High sigma-1 receptor binding may, however, limit its future application as a PET probe for imaging the NR2B subunit-containing NMDAr. Anaesthesia has an effect on NMDAr function and therefore can complicate interpretation of preclinical in vivo results. In addition, effects of endogenous compounds cannot be excluded. Despite these potential limitations, further studies are warranted to investigate the values of [(11)C]4 as an NR2B PET ligand.

Synthesis, radiolabeling and preclinical evaluation of a [11C]GMOM derivative as PET radiotracer for the ion channel of the N-methyl-D-aspartate receptor

INTRODUCTION Presently available PET ligands for the NMDAr ion channel generally suffer from fast metabolism. The purpose of this study was to develop a metabolically more stable ligand for the NMDAr ion channel, taking [11C]GMOM ([11C]1) as the lead compound. METHODS [11C]1, its fluoralkyl analogue [18F]PK209 ([18F]2) and the newly synthesized fluorovinyloxy analogue [11C]7b were evaluated ex vivo in male Wistar rats for metabolic stability. In addition, [11C]7b was subjected to a biodistribution study and its affinity (Ki) and lipophilicity (logD7.4) values were determined. RESULTS The addition of a vinyl chain in the fluoromethoxy moiety did not negatively alter the affinity of [11C]7b for the NMDAr, while lipophilicity was increased. Biodistribution studies showed higher uptake of [11C]7b in forebrain regions compared with cerebellum. Pre-treatment with MK-801 decreased the overall brain uptake significantly, but not in a region-specific manner. 45min after injection 78, 90 and 87% of activity in the brain was due to parent compound for [11C]1, [18F]2 and [11C]7b, respectively. In plasma, 26-31% of activity was due to parent compound. CONCLUSION Complete substitution of the alpha-carbon increased lipophilicity to more favorable values. Substitution of one or more hydrogens of the alpha-carbon atom in the methoxy moiety improved metabolic stability. In plasma, more parent compound was found for [18F]2 and [11C]7b then for [11C]1, although differences were not significant. At 45min, significantly more parent [18F]2 and [11C]7b was measured in the brain compared with [11C]1.

Subchronic treatment with phencyclidine in adolescence leads to impaired exploratory behavior in adult rats without altering social interaction or N-methyl-D-aspartate receptor binding levels

Although both the onset of schizophrenia and human phencyclidine (PCP) abuse typically present within the interval from adolescence to early adulthood, the majority of preclinical research employing the PCP model of schizophrenia has been conducted on neonatal or adult animals. The present study was designed to evaluate the behavioral and neurochemical sequelae of subchronic exposure to PCP in adolescence. Male 35–42‐day‐old Sprague Dawley rats were subcutaneously administered either saline (10 ml · kg−1) or PCP hydrochloride (10 mg · kg−1) once daily for a period of 14 days (n = 6/group). The animals were allowed to withdraw from treatment for 2 weeks, and their social and exploratory behaviors were subsequently assessed in adulthood by using the social interaction test. To examine the effects of adolescent PCP administration on the regulation of N‐methyl‐D‐aspartate receptors (NMDARs), quantitative autoradiography was performed on brain sections of adult, control and PCP‐withdrawn rats by using 20 nM 3H‐MK‐801. Prior subchronic exposure to PCP in adolescence had no enduring effects on the reciprocal contact and noncontact social behavior of adult rats. Spontaneous rearing in response to the novel testing arena and time spent investigating its walls and floor were reduced in PCP‐withdrawn animals compared with control. The long‐term behavioral effects of PCP occurred in the absence of persistent deficits in spontaneous locomotion or self‐grooming activity and were not mediated by altered NMDAR density. Our results document differential effects of adolescent PCP administration on the social and exploratory behaviors of adult rats, suggesting that distinct neurobiological mechanisms are involved in mediating these behaviors.

Serotonin augmentation therapy by escitalopram has minimal effects on amyloid-β levels in early-stage Alzheimer’s-like disease in mice

BackgroundDysfunction of the serotonergic (5-HTergic) system has been implicated in the cognitive and behavioural symptoms of Alzheimer’s disease (AD). Accumulation of toxic amyloid-β (Aβ) species is a hallmark of AD and an instigator of pathology. Serotonin (5-HT) augmentation therapy by treatment with selective serotonin reuptake inhibitors (SSRIs) in patients with AD has had mixed success in improving cognitive function, whereas SSRI administration to mice with AD-like disease has been shown to reduce Aβ pathology. The objective of this study was to investigate whether an increase in extracellular levels of 5-HT induced by chronic SSRI treatment reduces Aβ pathology and whether 5-HTergic deafferentation of the cerebral cortex could worsen Aβ pathology in the APPswe/PS1ΔE9 (APP/PS1) mouse model of AD.MethodsWe administered a therapeutic dose of the SSRI escitalopram (5 mg/kg/day) in the drinking water of 3-month-old APP/PS1 mice to increase levels of 5-HT, and we performed intracerebroventricular injections of the neurotoxin 5,7-dihydroxytryptamine (DHT) to remove 5-HTergic afferents. We validated the effectiveness of these interventions by serotonin transporter autoradiography (neocortex 79.7 ± 7.6%) and by high-performance liquid chromatography for 5-HT (neocortex 64% reduction). After 6 months of escitalopram treatment or housing after DHT-induced lesion, we evaluated brain tissue by mesoscale multiplex analysis and sections by IHC analysis.ResultsAmyloid-β-containing plaques had formed in the neocortex and hippocampus of 9-month-old APP/PS1 mice after 6 months of escitalopram treatment and 5-HTergic deafferentation. Unexpectedly, levels of insoluble Aβ42 were unaffected in the neocortex and hippocampus after both types of interventions. Levels of insoluble Aβ40 increased in the neocortex of SSRI-treated mice compared with those treated with vehicle control, but they were unaffected in the hippocampus. 5-HTergic deafferentation was without effect on the levels of insoluble/soluble Aβ42 and Aβ40 in both the neocortex and hippocampus. However, levels of soluble amyloid precursor protein α were reduced in the neocortex after 5-HTergic deafferentation.ConclusionsBecause this study shows that modulation of the 5-HTergic system has either no effect or increases levels of insoluble/soluble Aβ42 and Aβ40 in the cerebral cortex of APP/PS1 mice, our observations do not support 5-HT augmentation therapy as a preventive strategy for reducing Aβ pathology.