Introduction: Metabolic dysfunction is now recognized as a pivotal component of Alzheimer's disea... more Introduction: Metabolic dysfunction is now recognized as a pivotal component of Alzheimer's disease (AD), the most common dementia worldwide. However, the precise molecular mechanisms linking metabolic dysfunction to AD remain elusive. Objective: Here, we investigated the direct impact of soluble oligomeric amyloid beta (Aβ) peptides, the main molecular hallmark of AD, on the leptin system, a major component of central energy metabolism regulation. Methods: We developed a new time-resolved fluorescence resonance energy transfer-based Aβ binding assay for the leptin receptor (LepR) and studied the effect of Aβ on LepR function in several in vitro assays. The in vivo effect of Aβ on LepR function was studied in an Aβ-specific AD mouse model and in pro-opiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus. Results: We revealed specific and high-affinity (K i = 0.1 nM) binding of Aβ to LepR. Pharmacological characterization of this interaction showed that Aβ binds allosterically to the extracellular domain of LepR and negatively affects receptor function. Negative allosteric modulation of LepR by Aβ was detected at the level of signaling pathways (STAT-3, AKT, and ERK) in vitro and in vivo. Importantly, the leptin-induced response of POMC neurons, key players in the regulation of metabolic function, was completely abolished in the presence of Aβ. Conclusion: Our data indicate that Aβ is a negative allosteric modulator of LepR, resulting in impaired leptin action, and qualify LepR as a new and direct target of Aβ oligomers. Preventing the interaction of Aβ with LepR might improve both the metabolic and cognitive dysfunctions in AD condition.
In recent years, researchers have showed a growing interest in the mechanistic relationship betwe... more In recent years, researchers have showed a growing interest in the mechanistic relationship between bone marrow adipose tissue and adjacent tumors. However, the impact of bone marrow adipocytes on development of hematological malignancies, particularly multiple myeloma is unknown. With aging, bone marrow changes occur and fatty deposits can occupy up to 70% of the BM cavity. Interactions of bone marrow adipose tissue with bone cells and other immune cells, possibly suggest indirect ways in which bone marrow adipocytes may affect MM disease progression. Leptin, an adipokine released by adipocytes and crucial in energy homeostasis, displays immune modulatory properties but its role in anti-tumor immunity remains unclear. In this study we aimed to investigate the intriguing relationship between leptin receptor activation and invariant natural killer T (iNKT) cell mediated anti-tumor immunity in multiple myeloma. The murine immunocompetent 5T33MM model, mimicking the human disease close...
Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF)... more Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity-on-Target cytokines (AcTakines) are a novel class of immunocytokines with improved therapeutic index. A TNF-based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo. Upregulation of adhesion markers supports enhanced T-cell infiltration leading to control or elimination of solid tumors by, respectively, CAR T cells or a combination therapy with CD8-targeted type I interferon AcTakine. Cotreatment with a CD13-targeted type II interferon AcTakine leads to very rapid destruction of the tumor neovasculature and complete regression of large, established tumors. As no tumor markers are needed, safe and efficacious elimination of a broad range of tumor types becomes feasible.
Under normal physiological conditions, leptin and the leptin receptor (ObR) regulate the body wei... more Under normal physiological conditions, leptin and the leptin receptor (ObR) regulate the body weight by balancing food intake and energy expenditure. However, this adipocyte-derived hormone also directs peripheral processes, including immunity, reproduction, and bone metabolism. Leptin, therefore, can act as a metabolic switch connecting the body's nutritional status to high energy consuming processes. We provide an extensive overview of current structural insights on the leptin-ObR interface and ObR activation, coupling to signaling pathways and their negative regulation, and leptin functioning under normal and pathophysiological conditions (obesity, autoimmunity, cancer, …). We also discuss possible cross-talk with other receptor systems on the receptor (extracellular) and signaling cascade (intracellular) levels.
Interleukin 5 (IL-5) is the key cytokine in an eosinophil&amp... more Interleukin 5 (IL-5) is the key cytokine in an eosinophil's life span: it supports eosinophilopoiesis and eosinophil differentiation, contributes to eosinophil migration, tissue localisation and function, and prevents eosinophil apoptosis. Given the likely role of eosinophils in chronic inflammatory diseases, a lot of research over the past decade was aimed at antagonising IL-5 function. It appears from recent studies that, although this can easily be achieved in vitro, blocking IL-5 function in vivo is much more difficult than originally anticipated. Here, we review the current status of IL-5 and IL-5 receptor research, with emphasis on strategies to interfere with IL-5 function.
The leptin receptor (LR), a member of the class I cytokine receptor family, is composed of a sing... more The leptin receptor (LR), a member of the class I cytokine receptor family, is composed of a single subunit. Its extracellular domain consists of two so-called cytokine receptor homology domains, separated by an Ig-like domain, and two additional fibronectin type III modules. Requirements for LR activation were examined using a complementation strategy. Two LR mutants, LR-FFY-⌬box 1 and LR-F3, deficient in Janus kinase or signal transducer and activator of transcription (STAT) activation, respectively, were only able to generate a STAT3-dependent signal when coexpressed. Based on the requirements for Janus kinase/STAT
After its discovery in 1994, it soon became clear that leptin acts as an adipocyte-derived hormon... more After its discovery in 1994, it soon became clear that leptin acts as an adipocyte-derived hormone with a central role in the control of body weight and energy homeostasis. However, a growing body of evidence has revealed that leptin is a pleiotropic cytokine with activities on many peripheral cell types. Inappropriate leptin signaling can promote autoimmunity, certain cardiovascular diseases, elevated blood pressure and cancer, which makes leptin and the leptin receptor interesting targets for antagonism. Profound insights in the leptin receptor (LR) activation mechanisms are a prerequisite for the rational design of these antagonists. In this review, we focus on the molecular mechanisms underlying leptin receptor activation and signaling. We also discuss the current strategies to interfere with leptin signaling and their therapeutic potential.
Hemangioblastomas express erythropoietin and the patients often present with polycythemia. Serum ... more Hemangioblastomas express erythropoietin and the patients often present with polycythemia. Serum erythropoietin was measured using a commercial immunoassay, a functional erythropoietin assay and iso-electric focusing. Despite the polycythemia, serum erythropoietin remained low, while a functional erythropoietin-assay showed a 4-5 higher activity in serum compared to the immunoassay. Iso-electric focusing of serum erythropoietin indicated overrepresentation of highly sialylated erythropoietin isoforms produced by the tumor. As a result, altered affinity of the monoclonal antibody used in the immunoassay for the hypersialylated isoforms was suggested. Analysis of erythropoietin isoforms may be helpful in distinguishing the ectopic erythropoietin isoforms from normally glycosylated erythropoietin.
We have designed a sensitive and versatile bioassay for quantification of series of cytokines. Th... more We have designed a sensitive and versatile bioassay for quantification of series of cytokines. The assay makes use of chimeric receptors composed of the extracellular, ligand-binding part of the cognate cytokine receptor and the transmembrane and cytosolic part of the type I interferon receptor. Receptors can be homo- (e.g. erythropoietin), di- (e.g. interleukin-5), or even trimeric (e.g. interleukin-2). Stable expression of these chimeras in the 2fTGH cell line allows an interferon-type signaling, which makes a positive selection in conditioned medium possible or a negative selection using a toxic guanine analog. The cytokine of interest is quantified by the extent of cell survival or cell toxicity respectively, which can be measured by easy and cheap crystal violet staining. This bioassay is sensitive in the lower picogram per milliliter range and, in contrast to ELISA methods, only measures the concentration of biologically active cytokines. Using this approach, hypersensitive 2fTGH cell lines have been developed for type I and II interferons, erythropoietin, interleukin-2, and interleukin-5.
Leptin plays a central role in the control of body weight and energy homeostasis, but is a pleiot... more Leptin plays a central role in the control of body weight and energy homeostasis, but is a pleiotropic cytokine with activities on many peripheral cell types. In this review, we discuss the interaction of leptin with its receptor, and focus on the structural and mechanistic aspects of the extracellular aspects of leptin receptor (LR) activation. We provide an extensive overview of all structural information that has been obtained for leptin and its receptor via X-ray crystallography, electron microscopy, small-angle X-ray scattering, homology modeling, and mutagenesis studies. The available knowledge is integrated into putative models toward a recapitulation of the LR activation mechanism.
The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolis... more The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. We here provide genetic and biochemical evidence that the metabolic and immune functions of leptin can be uncoupled at the receptor level. First, homozygous mutant fatt/fatt mice carry a spontaneous splice mutation causing deletion of the leptin receptor (LR) immunoglobulin-like domain (IGD) in all LR isoforms. These mice are hyperphagic and morbidly obese, but display only minimal changes in size and cellularity of the thymus, and cellular immune responses are unaffected. These animals also displayed liver damage in response to concavalin A comparable to wild-type and heterozygous littermates. Second, treatment of healthy mice with a neutralizing nanobody targeting IGD induced weight gain and hyperinsulinaemia, but completely failed to block development of experimentally induced autoimmune diseases. These data indicate that leptin receptor deficiency or antagonism profoundly affects metabolism, with little concomitant effects on immune functions.
Leptin is a multifunctional hormone produced by the ob gene and is secreted by adipocytes that re... more Leptin is a multifunctional hormone produced by the ob gene and is secreted by adipocytes that regulate food intake and energy metabolism. Numerous studies demonstrated that leptin is a novel neuroprotective effector, however, the mechanisms are largely unknown. Herein, we demonstrate the protective activities of leptin after ischemic stroke and provide the first evidence for the involvement of the connexin 43 (Cx43) in leptin-mediated neuroprotection. We found that leptin treatment reduces the infarct volume, improves animal behavioral parameters, and inhibits the elevation of Cx43 expression in vivo. In vitro, leptin reverses ischemia-induced SY5Y and U87 cells Cx43 elevation, secreted glutamate levels in medium and SY5Y cell death, these roles could be abolished by leptin receptor blocker. Additionally, leptin administration upregulated the extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation. Moreover, ERK1/2 inhibitors pretreatment reversed the effects of leptin on...
Introduction: Metabolic dysfunction is now recognized as a pivotal component of Alzheimer's disea... more Introduction: Metabolic dysfunction is now recognized as a pivotal component of Alzheimer's disease (AD), the most common dementia worldwide. However, the precise molecular mechanisms linking metabolic dysfunction to AD remain elusive. Objective: Here, we investigated the direct impact of soluble oligomeric amyloid beta (Aβ) peptides, the main molecular hallmark of AD, on the leptin system, a major component of central energy metabolism regulation. Methods: We developed a new time-resolved fluorescence resonance energy transfer-based Aβ binding assay for the leptin receptor (LepR) and studied the effect of Aβ on LepR function in several in vitro assays. The in vivo effect of Aβ on LepR function was studied in an Aβ-specific AD mouse model and in pro-opiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus. Results: We revealed specific and high-affinity (K i = 0.1 nM) binding of Aβ to LepR. Pharmacological characterization of this interaction showed that Aβ binds allosterically to the extracellular domain of LepR and negatively affects receptor function. Negative allosteric modulation of LepR by Aβ was detected at the level of signaling pathways (STAT-3, AKT, and ERK) in vitro and in vivo. Importantly, the leptin-induced response of POMC neurons, key players in the regulation of metabolic function, was completely abolished in the presence of Aβ. Conclusion: Our data indicate that Aβ is a negative allosteric modulator of LepR, resulting in impaired leptin action, and qualify LepR as a new and direct target of Aβ oligomers. Preventing the interaction of Aβ with LepR might improve both the metabolic and cognitive dysfunctions in AD condition.
In recent years, researchers have showed a growing interest in the mechanistic relationship betwe... more In recent years, researchers have showed a growing interest in the mechanistic relationship between bone marrow adipose tissue and adjacent tumors. However, the impact of bone marrow adipocytes on development of hematological malignancies, particularly multiple myeloma is unknown. With aging, bone marrow changes occur and fatty deposits can occupy up to 70% of the BM cavity. Interactions of bone marrow adipose tissue with bone cells and other immune cells, possibly suggest indirect ways in which bone marrow adipocytes may affect MM disease progression. Leptin, an adipokine released by adipocytes and crucial in energy homeostasis, displays immune modulatory properties but its role in anti-tumor immunity remains unclear. In this study we aimed to investigate the intriguing relationship between leptin receptor activation and invariant natural killer T (iNKT) cell mediated anti-tumor immunity in multiple myeloma. The murine immunocompetent 5T33MM model, mimicking the human disease close...
Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF)... more Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity-on-Target cytokines (AcTakines) are a novel class of immunocytokines with improved therapeutic index. A TNF-based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo. Upregulation of adhesion markers supports enhanced T-cell infiltration leading to control or elimination of solid tumors by, respectively, CAR T cells or a combination therapy with CD8-targeted type I interferon AcTakine. Cotreatment with a CD13-targeted type II interferon AcTakine leads to very rapid destruction of the tumor neovasculature and complete regression of large, established tumors. As no tumor markers are needed, safe and efficacious elimination of a broad range of tumor types becomes feasible.
Under normal physiological conditions, leptin and the leptin receptor (ObR) regulate the body wei... more Under normal physiological conditions, leptin and the leptin receptor (ObR) regulate the body weight by balancing food intake and energy expenditure. However, this adipocyte-derived hormone also directs peripheral processes, including immunity, reproduction, and bone metabolism. Leptin, therefore, can act as a metabolic switch connecting the body's nutritional status to high energy consuming processes. We provide an extensive overview of current structural insights on the leptin-ObR interface and ObR activation, coupling to signaling pathways and their negative regulation, and leptin functioning under normal and pathophysiological conditions (obesity, autoimmunity, cancer, …). We also discuss possible cross-talk with other receptor systems on the receptor (extracellular) and signaling cascade (intracellular) levels.
Interleukin 5 (IL-5) is the key cytokine in an eosinophil&amp... more Interleukin 5 (IL-5) is the key cytokine in an eosinophil's life span: it supports eosinophilopoiesis and eosinophil differentiation, contributes to eosinophil migration, tissue localisation and function, and prevents eosinophil apoptosis. Given the likely role of eosinophils in chronic inflammatory diseases, a lot of research over the past decade was aimed at antagonising IL-5 function. It appears from recent studies that, although this can easily be achieved in vitro, blocking IL-5 function in vivo is much more difficult than originally anticipated. Here, we review the current status of IL-5 and IL-5 receptor research, with emphasis on strategies to interfere with IL-5 function.
The leptin receptor (LR), a member of the class I cytokine receptor family, is composed of a sing... more The leptin receptor (LR), a member of the class I cytokine receptor family, is composed of a single subunit. Its extracellular domain consists of two so-called cytokine receptor homology domains, separated by an Ig-like domain, and two additional fibronectin type III modules. Requirements for LR activation were examined using a complementation strategy. Two LR mutants, LR-FFY-⌬box 1 and LR-F3, deficient in Janus kinase or signal transducer and activator of transcription (STAT) activation, respectively, were only able to generate a STAT3-dependent signal when coexpressed. Based on the requirements for Janus kinase/STAT
After its discovery in 1994, it soon became clear that leptin acts as an adipocyte-derived hormon... more After its discovery in 1994, it soon became clear that leptin acts as an adipocyte-derived hormone with a central role in the control of body weight and energy homeostasis. However, a growing body of evidence has revealed that leptin is a pleiotropic cytokine with activities on many peripheral cell types. Inappropriate leptin signaling can promote autoimmunity, certain cardiovascular diseases, elevated blood pressure and cancer, which makes leptin and the leptin receptor interesting targets for antagonism. Profound insights in the leptin receptor (LR) activation mechanisms are a prerequisite for the rational design of these antagonists. In this review, we focus on the molecular mechanisms underlying leptin receptor activation and signaling. We also discuss the current strategies to interfere with leptin signaling and their therapeutic potential.
Hemangioblastomas express erythropoietin and the patients often present with polycythemia. Serum ... more Hemangioblastomas express erythropoietin and the patients often present with polycythemia. Serum erythropoietin was measured using a commercial immunoassay, a functional erythropoietin assay and iso-electric focusing. Despite the polycythemia, serum erythropoietin remained low, while a functional erythropoietin-assay showed a 4-5 higher activity in serum compared to the immunoassay. Iso-electric focusing of serum erythropoietin indicated overrepresentation of highly sialylated erythropoietin isoforms produced by the tumor. As a result, altered affinity of the monoclonal antibody used in the immunoassay for the hypersialylated isoforms was suggested. Analysis of erythropoietin isoforms may be helpful in distinguishing the ectopic erythropoietin isoforms from normally glycosylated erythropoietin.
We have designed a sensitive and versatile bioassay for quantification of series of cytokines. Th... more We have designed a sensitive and versatile bioassay for quantification of series of cytokines. The assay makes use of chimeric receptors composed of the extracellular, ligand-binding part of the cognate cytokine receptor and the transmembrane and cytosolic part of the type I interferon receptor. Receptors can be homo- (e.g. erythropoietin), di- (e.g. interleukin-5), or even trimeric (e.g. interleukin-2). Stable expression of these chimeras in the 2fTGH cell line allows an interferon-type signaling, which makes a positive selection in conditioned medium possible or a negative selection using a toxic guanine analog. The cytokine of interest is quantified by the extent of cell survival or cell toxicity respectively, which can be measured by easy and cheap crystal violet staining. This bioassay is sensitive in the lower picogram per milliliter range and, in contrast to ELISA methods, only measures the concentration of biologically active cytokines. Using this approach, hypersensitive 2fTGH cell lines have been developed for type I and II interferons, erythropoietin, interleukin-2, and interleukin-5.
Leptin plays a central role in the control of body weight and energy homeostasis, but is a pleiot... more Leptin plays a central role in the control of body weight and energy homeostasis, but is a pleiotropic cytokine with activities on many peripheral cell types. In this review, we discuss the interaction of leptin with its receptor, and focus on the structural and mechanistic aspects of the extracellular aspects of leptin receptor (LR) activation. We provide an extensive overview of all structural information that has been obtained for leptin and its receptor via X-ray crystallography, electron microscopy, small-angle X-ray scattering, homology modeling, and mutagenesis studies. The available knowledge is integrated into putative models toward a recapitulation of the LR activation mechanism.
The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolis... more The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. We here provide genetic and biochemical evidence that the metabolic and immune functions of leptin can be uncoupled at the receptor level. First, homozygous mutant fatt/fatt mice carry a spontaneous splice mutation causing deletion of the leptin receptor (LR) immunoglobulin-like domain (IGD) in all LR isoforms. These mice are hyperphagic and morbidly obese, but display only minimal changes in size and cellularity of the thymus, and cellular immune responses are unaffected. These animals also displayed liver damage in response to concavalin A comparable to wild-type and heterozygous littermates. Second, treatment of healthy mice with a neutralizing nanobody targeting IGD induced weight gain and hyperinsulinaemia, but completely failed to block development of experimentally induced autoimmune diseases. These data indicate that leptin receptor deficiency or antagonism profoundly affects metabolism, with little concomitant effects on immune functions.
Leptin is a multifunctional hormone produced by the ob gene and is secreted by adipocytes that re... more Leptin is a multifunctional hormone produced by the ob gene and is secreted by adipocytes that regulate food intake and energy metabolism. Numerous studies demonstrated that leptin is a novel neuroprotective effector, however, the mechanisms are largely unknown. Herein, we demonstrate the protective activities of leptin after ischemic stroke and provide the first evidence for the involvement of the connexin 43 (Cx43) in leptin-mediated neuroprotection. We found that leptin treatment reduces the infarct volume, improves animal behavioral parameters, and inhibits the elevation of Cx43 expression in vivo. In vitro, leptin reverses ischemia-induced SY5Y and U87 cells Cx43 elevation, secreted glutamate levels in medium and SY5Y cell death, these roles could be abolished by leptin receptor blocker. Additionally, leptin administration upregulated the extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation. Moreover, ERK1/2 inhibitors pretreatment reversed the effects of leptin on...
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Papers by Lennart Zabeau