Winnie Deuther-Conrad

Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of β-adrenergic receptors. Because BAT therapies based on cold exposure or β-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells—so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine–A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

Advanced glycation endproducts co-localize with inducible nitric oxide synthase in Alzheimer’s disease

Advanced glycation endproducts (AGEs), protein-bound oxidation products of sugars, have been shown to be involved in the pathophysiological processes of Alzheimers disease (AD). AGEs induce the expression of various pro-inflammatory cytokines and the inducible nitric oxide synthase (iNOS) leading to a state of oxidative stress. AGE modification and resulting crosslinking of protein deposits such as amyloid plaques may contribute to the oxidative stress occurring in AD. The aim of this study was to immunohistochemically compare the localization of AGEs and beta-amyloid (Abeta) with iNOS in the temporal cortex (Area 22) of normal and AD brains. In aged normal individuals as well as early stage AD brains (i.e. no pathological findings in isocortical areas), a few astrocytes showed co-localization of AGE and iNOS in the upper neuronal layers, compared with no astrocytes detected in young controls. In late AD brains, there was a much denser accumulation of astrocytes co-localized with AGE and iNOS in the deeper and particularly upper neuronal layers. Also, numerous neurons with diffuse AGE but not iNOS reactivity and some AGE and iNOS-positive microglia were demonstrated, compared with only a few AGE-reactive neurons and no microglia in controls. Finally, astrocytes co-localized with AGE and iNOS as well as AGE and were found surrounding mature but not diffuse amyloid plaques in the AD brain. Our results show that AGE-positive astrocytes and microglia in the AD brain express iNOS and support the evidence of an AGE-induced oxidative stress occurring in the vicinity of the characteristic lesions of AD. Hence activation of microglia and astrocytes by AGEs with subsequent oxidative stress and cytokine release may be an important progression factor in AD.

β‐Amyloid peptide potentiates inflammatory responses induced by lipopolysaccharide, interferon ‐γ and ‘advanced glycation endproducts’ in a murine microglia cell line

β‐Amyloid (Aβ) plaques are characteristic hallmarks of Alzheimers disease (AD). In AD, it has been suggested that activation of microglial cells might be the link between Aβ deposition and neuronal degeneration. Activated microglia are associated with senile plaques and produce free radicals and inflammatory cytokines. However, it is still not clear whether Aβ needs a prestimulated environment to exert its proinflammatory potential. Advanced glycation endproducts (AGEs), protein‐bound oxidation products of sugars, have been shown to accumulate in senile plaques and could induce a silent but chronic inflammation in the AD brain. We tested whether Aβ acts as an amplifier of a submaximal proinflammatory response initiated by exposure to chicken egg albumin‐AGE, lipopolysaccharide or interferon‐γ. Synthetic Aβ was used to produce three different samples (Aβ‐fibrilar; Aβ‐aggregated; Aβ‐AGE), which were characterized for β‐sheeted fibrils by the thioflavin‐T test and electron microscopy. As markers of microglial activation, nitric oxide, interleukin‐6, macrophage‐colony stimulation factor and tumour necrosis factor‐α production was measured. All three Aβ samples alone could not induce a detectable microglial response. The combination of Aβ preparations, however, with the coinducers provoked a strong microglial response, whereby Aβ‐AGE and fibrilar Aβ were more potent inflammatory signals than aggregated Aβ. Thus, Aβ in senile plaques can amplify microglial activation by a coexisting submaximal inflammatory stimulus. Hence, anti‐inflammatory therapeutics could either target the primary proinflammatory signal (e.g. by limiting AGE‐formation by AGE inhibitors or cross‐link breakers) or the amplifyer Aβ (e.g. by limiting Aβ production by β‐ or γ‐secretase inhibitors).

Signal transduction pathways in mouse microglia N-11 cells activated by advanced glycation endproducts (AGEs)

Deposition of cross‐linked insoluble protein aggregates such as amyloid plaques is characteristic for Alzheimers disease. Microglial activation by these extracullar deposits has been proposed to play a crucial role in functional degeneration as well as cell death of neurones. A sugar‐derived post‐translational modification of long‐lived proteins, advanced glycation endproducts (AGEs), activate specific signal transduction pathways, resulting in the up‐regulation of various pro‐inflammatory signals such as cytokines [interleukin‐6 (IL‐6), tumour necrosis factor‐alpha (TNF‐α)] and inducible nitric oxide synthase (iNOS). Our goal was to study AGE‐activated signal transduction pathways involved in the induction of pro‐inflammatory effectors in the murine microglial cell line N‐11. Chicken egg albumin‐AGE (CEA‐AGE), used as model AGE, induces nitric oxide (NO), TNF‐α and IL‐6 production. The AGE receptor, RAGE, and the transcription factor, nuclear factor kappa B (NF‐κB), appear to be involved in all pathways, since a neutralizing RAGE antibody and a peptide inhibiting NF‐κB translocation down‐regulated NO, TNF‐α and IL‐6 production. NO and TNF‐α, but not IL‐6 production appear to be regulated independently, since NOS inhibitors did not decrease TNF‐α secretion and a neutralizing TNF‐α antibody did not reduce NO production, while employment of NOS inhibitors reduced significantly the secretion of IL‐6. Inhibition of the MAP‐kinase‐kinase (MEK) and phosphatidylinositol 3‐kinase (PI3K) pathway, but not that of mitogen‐activated protein kinase‐p38 (MAPK‐p38), reduced NO, TNF‐α and IL‐6 significantly, suggesting that simultaneous activation of the first two pathways is necessary for the AGE‐induced induction of these pro‐inflammatory stimuli.

Glycoxidative stress creates a vicious cycle of neurodegeneration in Alzheimer’s disease — a target for neuroprotective treatment strategies?

Accumulation of Advanced Glycation Endproducts (AGEs) in the brain is a feature of ageing and degeneration, especially in Alzheimers disease (AD). Increased AGE levels explain many of the neuropathological and biochemical features of AD such as extensive protein crosslinking (beta-amyloid and MAP-tau), glial activation, oxidative stress and neuronal cell death. Oxidative stress and AGEs initiate a positive feedback loop, where normal age-related changes develop into a pathophysiological cascade. Combined intervention using antioxidants, anti-inflammatory drugs and AGE-inhibitors may be a promising neuroprotective strategy.

Cannabinoid receptor type 2 (CB2)-selective N-aryl-oxadiazolyl-propionamides: synthesis, radiolabelling, molecular modelling and biological evaluation

Background The endocannabinoid system is involved in many physiological and pathological processes. Two receptors (cannabinoid receptor type 1 (CB1) and type 2 (CB2)) are known so far. Many unwanted psychotic side effects of inhibitors of this system can be addressed to the interaction with CB1. While CB1 is one of the most abundant neuroreceptors, CB2 is expressed in the brain only at very low levels. Thus, highly potent and selective compounds for CB2 are desired. N-aryl-((hetero)aromatic)-oxadiazolyl-propionamides represent a promising class of such selective ligands for the human CB2. Here, a library of various derivatives is studied for suitable routes for labelling with 18F. Such 18F-labelled compounds can then be employed as CB2-selective radiotracers for molecular imaging studies employing positron emission tomography (PET). Results By varying the N-arylamide substructure, we explored the binding pocket of the human CB2 receptor and identified 9-ethyl-9H-carbazole amide as the group with optimal size. Radioligand replacement experiments revealed that the modification of the (hetero)aromatic moiety in 3-position of the 1,2,4-oxadiazoles shows only moderate impact on affinity to CB2 but high impact on selectivity towards CB2 with respect to CB1. Further, we could show by autoradiography studies that the most promising compounds bind selectively on CB2 receptors in mouse spleen tissue. Molecular docking studies based on a novel three-dimensional structural model of the human CB2 receptor in its activated form indicate that the compounds bind with the N-arylamide substructure in the binding pocket. 18F labelling at the (hetero)aromatic moiety at the opposite site of the compounds via radiochemistry was carried out. Conclusions The synthesized CB2-selective compounds have high affinity towards CB2 and good selectivity against CB1. The introduction of labelling groups at the (hetero)aromatic moiety shows only moderate impact on CB2 affinity, indicating the introduction of potential labelling groups at this position as a promising approach to develop CB2-selective ligands suitable for molecular imaging with PET. The high affinity for human CB2 and selectivity against human CB1 of the herein presented compounds renders them as suitable candidates for molecular imaging studies.

Time-dependent alterations of cholinergic markers after experimental traumatic brain injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disability. Cognitive deficits are believed to be connected with impairments of the cholinergic system. The present study was conducted to evaluate the cholinergic system in a model of focal brain injury with special attention to the time course of posttraumatic events in critical brain regions. Three groups of male Sprague-Dawley rats (post-TBI survival time: 2 h, 24 h and 72 h) were subjected to sham-operation (control) or controlled cortical impact injury. Receptor densities were determined on frozen ipsilateral sagittal brain sections with [(3)H]epibatidine (nicotinic acetylcholine receptors) and [(3)H]QNB (muscarinic acetylcholine receptors). The density of the vesicular acetylcholine transporter (vAChT) was evaluated with (-)[(3)H]vesamicol. Compared to control, vAChT was lowered (up to 50%) at each time point after trauma, with reductions in olfactory tubercle, basal forebrain, motor cortex, putamen, thalamic and hypothalamic areas and the gigantocellular reticular nucleus. Time-dependent reductions of about 20% of nAChR-density in the thalamus, hypothalamus, olfactory tubercle, gigantocellular reticular nucleus and motor cortex were observed post-TBI at 24 and 72 h. The same brain regions showed reductions of mAChR at 24 and 72 h after trauma with additional decreases in the corpus callosum, basal forebrain and anterior olfactory nucleus. In conclusion, cholinergic markers showed significant time-dependent impairments after TBI. Considering the role of the cholinergic system for cognitive processes in the brain, it seems likely that these impairments contribute to clinically relevant cognitive deficits.

Gastric Bypass Surgery Recruits a Gut PPAR-α-Striatal D1R Pathway to Reduce Fat Appetite in Obese Rats

Bariatric surgery remains the single most effective long-term treatment modality for morbid obesity, achieved mainly by lowering caloric intake through as yet ill-defined mechanisms. Here we show in rats that Roux-en-Y gastric bypass (RYGB)-like rerouting of ingested fat mobilizes lower small intestine production of the fat-satiety molecule oleoylethanolamide (OEA). This was associated with vagus nerve-driven increases in dorsal striatal dopamine release. We also demonstrate that RYGB upregulates striatal dopamine 1 receptor (D1R) expression specifically under high-fat diet feeding conditions. Mechanistically, interfering with local OEA, vagal, and dorsal striatal D1R signaling negated the beneficial effects of RYGB on fat intake and preferences. These findings delineate a molecular/systems pathway through which bariatric surgery improves feeding behavior and may aid in the development of novel weight loss strategies that similarly modify brain reward circuits compromised in obesity.

Protein „AGEing”– cytotoxicity of a glycated protein increases with its degree of AGE-modification

Summary Non-enzymatic glycation of proteins with reducing sugars and subsequent transition metal-catalyzed oxidations leads to the formation of protein-bound „advanced glycation endproducts” (AGEs). They accumulate on long-lived proteins including on and in the vicinity of the β-amyloid plaques in Alzheimer‘s disease (AD). Since the AGE modification of a protein increases with time, and such a „long-term incubation” might also occur in the AD brain, we investigated whether an increase in the cytotoxic effects of an AGE-modified model protein occurs over time. Bovine serum albumin (BSA) was modified by glucose for defined time periods, and the viability of SH-SY5Y neuroblastoma cells, incubated with the differentially AGE-modified BSA samples, was measured with the MTT assay. Cytotoxicity of the AGE-modified BSAs increased in correlation to the incubation time with glucose. Among the AGE-specific markers, browning (OD400) correlated best with cytotoxicity, followed by AGE-specific fluorescence and the defined AGE, carboxymethyllysine. Since AGEs accumulate in AD over time, they may be one of the „age-related” factors contributing to neuronal cell death in Alzheimer‘s disease.Zusammenfassung Die nicht-enzymatische Glykierung von Proteinen mit reduzierenden Zuckern und die nachfolgende Oxidation der frühen Reaktionsprodukte durch Übergangsmetalle führt zur Bildung von proteingebundenen „advanced glycation endproducts” (AGEs). AGEs akkumulieren auf langlebigen Proteinen und sind auch struktureller Bestandteil der β-Amyloidplaques bei der Alzheimerschen Demenz (AD). Das AGE-induzierte zytotoxische Potential eines Proteins ist wahrscheinlich direkt abhängig von dem Ausmaß der AGE-Modifikation. Da die AGE-Modifikation eines Proteins unter anderem von der Reaktionszeit bestimmt wird, könnte die lange Lebenszeit der β-Amyloidplaques im AD-Gehirn zu einer hohen lokalen AGE-Konzentration führen, die für den Krankheitsverlauf maßgeblich sein könnte. Bovines Serumalbumin (BSA) wurde durch 1- bis 7-wöchige Inkubation mit Glukose unterschiedlich stark AGE-modifiziert. Die Überlebensrate von SH-SY5Y Neuroblastom-Zellen wurde nach 18-stündiger Inkubation mit diesen verschiedenen AGE-BSAs durch den MTT-Assay bestimmt. Die Zytotoxizität des AGE-modifizierten BSAs erhöhte sich signifikant in Abhängigkeit vom AGE-Modifizierungsgrad, d.h. von der Dauer der Inkubation des BSA mit Glukose. Von den AGE-spezifischen Parametern korrelierte die „Bräunung“ (OD400nm) am besten mit der Zytotoxizität, gefolgt von der AGE-spezifischen Fluoreszenz und der Menge des strukturell definierten AGEs Carboxymethyllysin. Da AGEs in der AD mit der Dauer der Krankheit akkumulieren können, vermuten wir in den AGE-Strukturen einen der „altersbezogenen” Faktoren, die zum Verlust neuronaler Zellfunktionen in der Alzheimerschen Demenz beitragen.

Radiosynthesis and first evaluation in mice of [18F]NS14490 for molecular imaging of α7 nicotinic acetylcholine receptors

[(18)F]NS14490, a new potential radiotracer for neuroimaging of α7 nicotinic acetylcholine receptors (α7 nAChRs), was synthesized and evaluated in vitro and in vivo. Radioligand binding studies using [(3)H]methyllycaconitine and NS14490 as competitor showed a good target affinity (K(i,α7) = 2.5 nM) and a high selectivity towards other nAChRs. Radiosynthesis of [(18)F]NS14490 was performed by two different labelling procedures: a two-step synthesis using a prosthetic group, which led to 7% labelling yield, and the convenient direct nucleophilic substitution of the corresponding tosylate precursor, which resulted in 70% labelling yield. After optimisation of the isolation, purification and formulation process, biodistribution studies were performed in CD-1 mice. The brain uptake of [(18)F]NS14490 was comparably low (0.16% ID g(-1) wet weight at 5 min p.i.). The radiotracer showed a high metabolic stability in plasma and brain. Also, the target specificity was proven by pre-administration of a highly affine α7 ligand providing a rationale basis for further in vivo evaluation.