Metabotropic Glutamate Receptors – Regulation of Acute and Chronic Stress-Related Behavior and Physiology

Etiology and pharmacotherapy of stress-related psychiatric conditions and comorbid somatic pathologies are nowadays areas of high unmet medical need and intense research. It is widely accepted that stressors holding a chronic and psychosocial component represent the most acknowledged risk factor. The L-glutamatergic system represents the primary excitatory neurotransmitter system of the mammalian brain and mGlu receptors acting as important pre- and postsynaptic regulators of neurotransmission provide a mechanism by which fast synaptic responses through iGlu receptors can be fine-tuned. During the last decades, research on mGlu receptors advanced remarkably and much attention was given to the mGlu5 and mGlu7 subtypes in acute stress, fear and depression-related behavior and physiology (see Introduction). As the most widely distributed throughout the mammalian brain, the presynaptic mGlu7 receptor is an important regulator of glutamatergic function and postulated to be critical for both normal CNS functioning and a range of stress-related disorders. Although genetic and pharmacological approaches have helped to understand mGlu7’s function in a host of behavioral and physiological processes, available allosteric ligands have often yielded disparate results despite displaying similar pharmacological properties in vitro. The recent discovery of XAP044 raised great hope to resolve this discrepancy. XAP044 was characterized as the first mGlu7-selective full antagonist that blocks the receptor’s signaling pathways by binding to its large VFTD, but not to allosteric sites within the transmembrane domain. Thus, XAP044 presumably binds via a novel mechanism compared to that from known selective ligands. It was part of the present PhD thesis to characterize this novel compound XAP044 in vivo in a broad battery of acute stress tests for depression, fear and anxiety in mice. It was shown that XAP044 is systemically active and demonstrates a wide spectrum of anti-stress, antidepressant and anxiolytic-like efficacy, strongly supporting pharmacological blockade of mGlu7 as a promising mode of action for future treatment of stress-related disorders of emotion in man. Also the mGlu5 subtype has become a recent focus for drug discovery efforts (see Introduction). Due to its physical and functional association with the postsynaptic NMDA receptor the mGlu5 subtype is considered as a good target to modulate NMDA receptor function. This is an important consideration as, for instance, the NMDA receptor antagonist ketamine – despite showing rapid and sustained efficacy in clinical depression trials – is also associated with severe cognition-altering and dissociative effects. To date, several mGlu5 NAMs have been reported to have therapeutic potential for numerous conditions including clinical depression and anxiety disorders. A recent study revealed the mGlu5 NAM basimglurant as a promising antidepressant drug with the potential to also alleviate comorbidities such as anxiety and pain. Despite the overall well-established link between mGlu5 and mGlu7 and acute stress-related behavior and physiology, the roles these receptors play in chronic stress-related conditions is only little explored. The CSC paradigm represents a powerful animal model as it displays harmful behavioral, physiological and immunological changes induced by chronic psychosocial stress. Those consequences are relevant for the development of psychiatric, somatic and/or gastrointestinal disorders in humans and the question whether mGlu5 and mGlu7 have the potential to exert control on these pathological consequences is of great interest, and it may suggest future therapeutic strategies for the treatment of chronic stress-related disorders in humans, i.e. a wide clinical application spectrum. In a first step of the present PhD thesis, the molecular changes were assessed that occur within the mGlu receptor system in response to CSC exposure. Here, an increase by trend of mGlu5 mRNA was found in the hypothalamus. Additional saturation binding analysis revealed increased mGlu5 protein binding specifically in the hippocampus. Furthermore, robust downregulation of mGlu7 mRNA was found specifically in the PFC. In contrast, mGlu2 and mGlu3 were not dysregulated upon CSC exposure. Taken together, the present results indicate specific CSC-induced alterations of mGlu5 and mGlu7 expression in stress-sensitive brain regions involved in the regulation of behavior and HPA axis functionality, and thus provide early evidence towards a role of specific mGlu receptor subtypes in chronic psychosocial stress-induced pathophysiology. In a next step, the influence of genetic ablation of mGlu7 on behavioral, physiological and immunological consequences of CSC was analyzed to reveal the potential role of the endogenous mGlu7 receptor during chronic psychosocial stress. Indeed, genetic ablation of mGlu7 relieved multiple CSC induced alterations; mGlu7 deficient mice were protected against the CSC-induced anxiety-prone phenotype as well as against several CSC-induced physiological and immunological consequences such as HPA axis dysfunction and colonic inflammation, respectively. These findings point to a distinct role of mGlu7 in modulating a wide range of affective and somatic alteration that occur upon CSC exposure. Moreover, the stress-protective phenotype of genetic mGlu7 ablation suggests mGlu7 pharmacological blockade to be a possible treatment strategy for chronic stress-related emotional and somatic conditions in man. In the last part of the present thesis, the potentially beneficial role of genetic and pharmacological mGlu5 inhibition on CSC-induced alterations (the same broad range as presented above) was analyzed. Interestingly, also mGlu5 deficient mice were protected against a variety of CSC-induced physiological and behavioral changes, including the newly established CSC-induced increase in SIH response. Moreover, the effects of the mGlu5 NAM CTEP, a close analogue to the clinically active drug basimglurant with long half-life in rodents, were studied on a wider range of CSC-affected parameters. Here, CTEP relieved in a dose-dependent manner various CSC-induced consequences such as HPA axis dysfunction, immunological alterations and colonic inflammation, suggesting that mGlu5 is a relevant mediator for a wide range of alterations induced by chronic psychosocial stress and a potentially valuable drug target for the treatment of stress-related somatic pathologies. In conclusion, the present PhD thesis provides clear evidence for the importance of especially the mGlu5 and mGlu7 subtypes in the regulation of acute and chronic stress-related behavior and physiology, lending further support towards future development of mGlu5- and mGlu7-selective antagonists and their administration as therapy for stress-related psychiatric and somatic disorders in humans

Metabotropic glutamate receptor subtype 7 controls maternal care, maternal motivation and maternal aggression in mice

The group III metabotropic glutamate receptor subtype 7 (mGlu7) is an important regulator of glutamatergic and GABAergic neurotransmission and known to mediate emotionality and male social behavior. However, a possible regulatory role in maternal behavior remains unknown to date. Adequate expression of maternal behavior is essential for successful rearing and healthy development of the young. By understanding genetic and neural mechanisms underlying this important prosocial behavior, we gain valuable insights into possible dysregulations. Using genetic ablation as well as pharmacological modulation, we studied various parameters of maternal behavior in two different mouse strains under the influence of mGlu7. We can clearly show a regulatory role of mGlu7 in maternal behavior. Naive virgin female C57BL/6 mGlu7 knockout mice showed more often nursing postures and less spontaneous maternal aggression compared to their heterozygous and wildtype littermates. In lactating C57BL/6 wildtype mice, acute central activation of mGlu7 by the selective agonist AMN082 reduced arched back nursing and accelerated pup retrieval without affecting maternal aggression. In addition, in lactating CD1 wildtype mice the selective mGlu7 antagonist XAP044 increased both pup retrieval and maternal aggression. With respect to receptor expression levels, mGlu7 mRNA expression was higher in lactating vs virgin C57BL/6 mice in the prefrontal cortex, but not hypothalamus or hippocampus. In conclusion, these findings highlight a significant role of the mGlu7 receptor subtype in mediating maternal behavior in mice. Region-dependent studies are warranted to further extend our knowledge on the specific function of the brain glutamate system in maternal behavior

Impact of vitamin D metabolism on clinical epigenetics

The bioactive vitamin D (VD) metabolite, 1,25-dihydroxyvitamin D3 regulates essential pathways of cellular metabolism and differentiation via its nuclear receptor (VDR). Molecular mechanisms which are known to play key roles in aging and cancer are mediated by complex processes involving epigenetic mechanisms contributing to efficiency of VD-activating CYP27A1 and CYP27B1 or inactivating CYP24 enzymes as well as VDR which binds to specific genomic sequences (VD response elements or VDREs). Activity of VDR can be modulated epigenetically by histone acetylation. It co-operates with other nuclear receptors which are influenced by histone acetyl transferases (HATs) as well as several types of histone deacetylases (HDACs). HDAC inhibitors (HDACi) and/or demethylating drugs may contribute to normalization of VD metabolism. Studies link VD signaling through the VDR directly to distinct molecular mechanisms of both HAT activity and the sirtuin class of HDACs (SIRT1) as well as the forkhead transcription factors thus contributing to elucidate complex epigenetic mechanisms for cancer preventive actions of VD

The Emerging Role of Metabotropic Glutamate Receptors in the Pathophysiology of Chronic Stress-Related Disorders

Chronic stress-related psychiatric conditions such as anxiety, depression, and alcohol abuse are an enormous public health concern. The etiology of these pathologies is complex, with psychosocial stressors being among the most frequently discussed risk factors. The brain glutamatergic neurotransmitter system has often been found involved in behaviors and pathophysiologies resulting from acute stress and fear. Despite this, relatively little is known about the role of glutamatergic system components in chronic psychosocial stress, neither in rodents nor in humans. Recently, drug discovery efforts at the metabotropic receptor subtypes of the glutamatergic system (mGlu1-8 receptors) led to the identification of pharmacological tools with emerging potential in psychiatric conditions. But again, the contribution of individual mGlu subtypes to the manifestation of physiological, molecular, and behavioral consequences of chronic psychosocial stress remains still largely unaddressed. The current review will describe animal models typically used to analyze acute and particularly chronic stress conditions, including models of psychosocial stress, and there we will discuss the emerging roles for mGlu receptor subtypes. Indeed, accumulating evidence indicates relevance and potential therapeutic usefulness of mGlu2/3 ligands and mGlu5 receptor antagonists in chronic stress-related disorders. In addition, a role for further mechanisms, e.g. mGlu7-selective compounds, is beginning to emerge. These mechanisms are important to be analyzed in chronic psychosocial stress paradigms, e.g. in the chronic subordinate colony housing (CSC) model. We summarize the early results and discuss necessary future investigations, especially for mGlu5 and mGlu7 receptor blockers, which might serve to suggest improved therapeutic strategies to treat stress-related disorders

Induction of Suppressor Cells and Increased Tumor Growth following Chronic Psychosocial Stress in Male Mice.

To study the impact of psychosocial stress on the immune system, male mice were subjected to chronic subordinate colony housing (CSC), a preclinically validated mouse model for chronic psychosocial stress. CSC substantially affected the cell composition of the bone marrow, blood, and spleen by inducing myelopoiesis and enhancing the frequency of regulatory T cells in the CD4 population. Expansion of the myeloid cell compartment was due to cells identified as immature inflammatory myeloid cells having the phenotype of myeloid-derived suppressor cells of either the granulocytic or the monocytic type. Catecholaminergic as well as TNF signaling were implicated in these CSC-induced cellular shifts. Although the frequency of regulatory cells was enhanced following CSC, the high capacity for inflammatory cytokine secretion of total splenocytes indicated an inflammatory immune status in CSC mice. Furthermore, CSC enhanced the suppressive activity of bone marrow-derived myeloid-derived suppressor cells towards proliferating T cells. In line with the occurrence of suppressor cell types such as regulatory T cells and myeloid-derived suppressor cells, transplanted syngeneic fibrosarcoma cells grew better in CSC mice than in controls, a process accompanied by pronounced angiogenesis and clustering of immature myeloid cells in the tumor tissue. In addition, tumor implantation after CSC reinforced the CSC-induced increase in myeloid-derived suppressor cells and regulatory T cell frequencies while the CSC-induced cellular changes eased off in mice without tumor. Together, our data suggest a role for suppressor cells such as regulatory T cells and myeloid-derived suppressor cells in the enhanced tumor growth after chronic psychosocial stress

Effect of CSC after 23 days of tumor growth.

<p>BFS1 tumor cells were inoculated subcutaneously after 19 days of either SHC (white bars, n = 7) or CSC (black bars, n = 8) and tumor growth was monitored for 23 days <b>(A). T</b>he weight of the tumor tissue was determined on day 23 after termination of CSC/SHC <b>(B)</b>. Immunohistochemical staining is shown for Gr1⁺ (green) and endothelial (Meca32, red) cells in tumor sections <b>(C)</b>. Percentages of CD11b⁺cells, CD11b⁺Ly6G⁺Ly6C^int cells (PMN-MDSC), and CD11b⁺Ly6G^-Ly6C^high cells (MO-MDSC) in spleens of individual mice were quantified (SHC: n = 7, total splenocytes: 4.9±1.7x10⁷, CSC: n = 8, total splenocytes: 5.6±0.7x10⁷) <b>(D)</b>. This experiment was performed once *p < 0.05; ***p < 0.001 (Student´s t-test).</p

Involvement of catecholamines, TNF, or TNFR2 on CSC-caused cellular shifts.

<p><b>(A)</b> Mice were treated either with PBS or 6-OH dopamine (6OHDA) before induction of CSC/SHC. Splenocytes from individual PBS-treated SHC (white bars, n = 4, total splenocytes: 3.4±0.6x10⁷) and CSC mice (black bars, n = 3, total splenocytes: 4.1±1.6x10⁷), 6OHDA-treated SHC (light grey bars, n = 4, total splenocytes: 4.1±0.7x10⁷) and CSC mice (dark grey bars, n = 3, total splenocytes: 6.3±1x10⁷) were isolated after 19d of SHC/CSC and analyzed by flow cytometry. The percentages of CD11b⁺ cells (SHC/PBS: 3.3±0.6x10⁶; CSC/PBS: 6.9±2.7x10⁶; SHC/6OHDA: 4.9±0.8x10⁶; CSC/6OHDA: 9.5±0.2x10⁶), CD11b⁺Ly6G⁺Ly6C^int (PMN-MDSC, SHC/PBS: 0.4±0.1x10⁶; CSC/PBS: 2.9±1.1x10⁶; SHC/6OHDA: 1.0±0.2x10⁶; CSC/6OHDA: 2.1±0.0x10⁶), and CD11b⁺Ly6G^-Ly6C^high (MO-MDSC, SHC/PBS: 0.2±0.03x10⁶; CSC/PBS: 0.6±0.2x10⁶; SHC/6OHDA: 0.5±0.1x10⁶; CSC/6OHDA: 1.2±0.02x10⁶) were determined. <b>(B)</b> Wild type (Bl/6) or TNF-deficient (TNF^-/-) mice were exposed to SHC/CSC. The percentages of CD11b⁺ cells, PMN-MDSC, and MO-MDSC of individual Bl/6-SHC- (white bars, n = 4, total splenocytes: 4.2±1.7x10⁷), Bl/6-CSC- (black bars, n = 3, total splenocytes: 8.9±2.4x10⁷), TNF^-/—SHC (light gray bars, n = 6, total splenocytes: 6.9±1.3x10⁷), and TNF^-/—CSC-mice (dark grey bars, n = 5, total splenocytes: 9.3±1.9x10⁷) were quantified. The percentages of CD11b⁺ cells (SHC/Bl/6: 4.0±1.6x10⁶; CSC/Bl/6: 16.1±4.0x10⁶; SHC/TNF^-/-: 6.4±1.4x10⁶; CSC/TNF^-/-: 10.8±2.4x10⁶), CD11b⁺Ly6G⁺Ly6C^int (PMN-MDSC, SHC/Bl/6: 1.2±0.5x10⁶; CSC/Bl/6: 6.0±1.5x10⁶; SHC/TNF^-/-: 1.4±0.3x10⁶; CSC/TNF^-/-: 2.9±0.1x10⁶), and CD11b⁺Ly6G^-Ly6C^high (MO-MDSC, SHC/Bl/6: 0.5±0.3x10⁶; CSC/Bl/6: 1.8±0.5x10⁶; SHC/TNF^-/-: 0.5±0.1x10⁶; CSC/TNF^-/-: 1.1±0.2x10⁶) were determined. <b>(C)</b> Wild type (Bl/6-SHC, n = 3, white bars, total splenocytes: 7.1±1.5x10⁷; Bl/6-CSC, n = 4, black bars, total splenocytes: 9.5±2.1x10⁷) or TNFR2-deficient (TNFR2^-/- SHC, n = 4, light gray bars, total splenocytes: 6.3±1.2x10⁷; TNFR2^-/- CSC, n = 4, dark grey bars, total splenocytes: 5.1±0.9x10⁷) mice were exposed to SHC/CSC. The percentages of CD11b⁺ cells (SHC/WT: 7.1±1.5x10⁶; CSC/WT: 17.8±3.9x10⁶; SHC/TNFR2^-/-: 5.9±1.1x10⁶; CSC/TNFR2^-/-: 6.9±1.2x10⁶), PMN-MDSC (SHC/WT: 2.0±0.4x10⁶; CSC/WT: 6.1±1.3x10⁶; SHC/TNFR2^-/-: 1.6±0.3x10⁶; CSC/TNFR2^-/-: 2.0±0.4x10⁶), and MO-MDSC (SHC/WT: 1.2±0.2x10⁶; CSC/WT: 3.3+0.7x10⁶; SHC/TNFR2^-/-: 0.9±0.2x10⁶; CSC/TNFR2^-/-: 0.8±0.14x10⁶)(left graph) or Treg cells (SHC/WT: 1.4±0.2x10⁶; CSC/WT: 2.0±0.4x10³; SHC/TNFR2^-/-: 1.0±0.2x10⁶; CSC/TNFR2^-/-: 0.6±0.1x10⁶)(right graph) were quantified. These experiments were performed twice. *p < 0.05; **p < 0.01; ***p < 0.001 (2way ANOVA).</p

Functional activity of bone marrow-derived PMN-MDSC and MO-MDSC after CSC.

<p>CD11b⁺Ly6G⁺Ly6C^int cells (PMN-MDSC) and CD11b⁺Ly6G^-Ly6C⁺ cells (MO-MDSC) were isolated from bone marrow (BM) or spleen of individual mice after 19d of CSC (black bars, n = 4) or SHC (white bars, n = 4) and stimulated with LPS + IFNγ followed by IL-10 <b>(A)</b> and NO₂^- <b>(B)</b> determination in the supernatants. MO-MDSC (CD11b⁺Ly6G^-Ly6C⁺) were isolated from the bone marrow of mice after 19d of CSC (black bars, n = 4) or SHC (white bars, n = 4) and co-cultured with proliferating splenocytes from naive mice. Relative proliferation of CD4⁺ (left graph) and CD8⁺ (right graph) T cells is shown at different T cell to MO-MDSC ratios <b>(C).</b> The NO₂^- contents of the supernatants from (C) were measured <b>(D)</b>. These experiments were performed once *p < 0.05; **p < 0.01; ***p < 0.001 (Student´s t-test).</p

Induction of immature myeloid cells by CSC.

<p>Splenocytes from CSC (black bars) and SHC (white bars) mice were isolated and analyzed by flow cytometry. The percentages of splenocytes from individual mice (SHC: n = 4, spleen cell pool: 1.7x10⁸; CSC: n = 4, spleen cell pool: 2.4x10⁸) <b>(A)</b>, of blood cells counted in 0.1ml of blood (SHC: n = 8, CSC: n = 8) <b>(B),</b> and of bone marrow cells (SHC: n = 3, CSC: n = 3) <b>(C)</b> are shown depending on their staining pattern for CD11b⁺Ly6G⁺Ly6C^int (PMN-MDSC), and CD11b⁺Ly6G^-Ly6C^high (MO-MDSC) (left graphs) or B220⁺ and CD3⁺ (right graphs). *p < 0.05; **p < 0.01; ***p < 0.001 (Student´s t-test). These experiments were performed more than 3 times.</p