Serotonin effects on human iPSC-derived neural cell functions: from mitochondria to depression.

Depression's link to serotonin dysregulation is well-known. The monoamine theory posits that depression results from impaired serotonin activity, leading to the development of antidepressants targeting serotonin levels. However, their limited efficacy suggests a more complex cause. Recent studies highlight mitochondria as key players in depression's pathophysiology. Mounting evidence indicates that mitochondrial dysfunction significantly correlates with major depressive disorder (MDD), underscoring its pivotal role in depression. Exploring the serotonin-mitochondrial connection, our study investigated the effects of chronic serotonin treatment on induced-pluripotent stem cell-derived astrocytes and neurons from healthy controls and two case study patients. One was a patient with antidepressant non-responding MDD ("Non-R") and another had a non-genetic mitochondrial disorder ("Mito"). The results revealed that serotonin altered the expression of genes related to mitochondrial function and dynamics in neurons and had an equalizing effect on calcium homeostasis in astrocytes, while ATP levels seemed increased. Serotonin significantly decreased cytosolic and mitochondrial calcium in neurons. Electrophysiological measurements evidenced that serotonin depolarized the resting membrane potential, increased both sodium and potassium current density and ultimately improved the overall excitability of neurons. Specifically, neurons from the Non-R patient appeared responsive to serotonin in vitro, which seemed to improve neurotransmission. While it is unclear how this translates to the systemic level and AD resistance mechanisms are not fully elucidated, our observations show that despite his treatment resistance, this patient's cortical neurons are responsive to serotonergic signals. In the Mito patient, evidence suggested that serotonin, by increasing excitability, exacerbated an existing hyperexcitability highlighting the importance of considering mitochondrial disorders in patients with MDD, and avoiding serotonin-increasing medication. Taken together, our findings suggested that serotonin positively affects calcium homeostasis in astrocytes and increases neuronal excitability. The latter effect must be considered carefully, as it could have beneficial or detrimental implications based on individual pathologies.

Mitochondrial and Cellular Function in Fibroblasts, Induced Neurons, and Astrocytes Derived from Case Study Patients: Insights into Major Depression as a Mitochondria-Associated Disease.

The link between mitochondria and major depressive disorder (MDD) is increasingly evident, underscored both by mitochondria's involvement in many mechanisms identified in depression and the high prevalence of MDD in individuals with mitochondrial disorders. Mitochondrial functions and energy metabolism are increasingly considered to be involved in MDD's pathogenesis. This study focused on cellular and mitochondrial (dys)function in two atypical cases: an antidepressant non-responding MDD patient ("Non-R") and another with an unexplained mitochondrial disorder ("Mito"). Skin biopsies from these patients and controls were used to generate various cell types, including astrocytes and neurons, and cellular and mitochondrial functions were analyzed. Similarities were observed between the Mito patient and a broader MDD cohort, including decreased respiration and mitochondrial function. Conversely, the Non-R patient exhibited increased respiratory rates, mitochondrial calcium, and resting membrane potential. In conclusion, the Non-R patient's data offered a new perspective on MDD, suggesting a detrimental imbalance in mitochondrial and cellular processes, rather than simply reduced functions. Meanwhile, the Mito patient's data revealed the extensive effects of mitochondrial dysfunctions on cellular functions, potentially highlighting new MDD-associated impairments. Together, these case studies enhance our comprehension of MDD.

Structure-function relationships of the disease-linked A218T oxytocin receptor variant.

Various single nucleotide polymorphisms (SNPs) in the oxytocin receptor (OXTR) gene have been associated with behavioral traits, autism spectrum disorder (ASD) and other diseases. The non-synonymous SNP rs4686302 results in the OXTR variant A218T and has been linked to core characteristics of ASD, trait empathy and preterm birth. However, the molecular and intracellular mechanisms underlying those associations are still elusive. Here, we uncovered the molecular and intracellular consequences of this mutation that may affect the psychological or behavioral outcome of oxytocin (OXT)-treatment regimens in clinical studies, and provide a mechanistic explanation for an altered receptor function. We created two monoclonal HEK293 cell lines, stably expressing either the wild-type or A218T OXTR. We detected an increased OXTR protein stability, accompanied by a shift in Ca2+ dynamics and reduced MAPK pathway activation in the A218T cells. Combined whole-genome and RNA sequencing analyses in OXT-treated cells revealed 7823 differentially regulated genes in A218T compared to wild-type cells, including 429 genes being associated with ASD. Furthermore, computational modeling provided a molecular basis for the observed change in OXTR stability suggesting that the OXTR mutation affects downstream events by altering receptor activation and signaling, in agreement with our in vitro results. In summary, our study provides the cellular mechanism that links the OXTR rs4686302 SNP with genetic dysregulations associated with aspects of ASD.

Induced neural progenitor cells and iPS-neurons from major depressive disorder patients show altered bioenergetics and electrophysiological properties.

The molecular pathomechanisms of major depressive disorder (MDD) are still not completely understood. Here, we follow the hypothesis, that mitochondria dysfunction which is inevitably associated with bioenergetic disbalance is a risk factor that contributes to the susceptibility of an individual to develop MDD. Thus, we investigated molecular mechanisms related to mitochondrial function in induced neuronal progenitor cells (NPCs) which were reprogrammed from fibroblasts of eight MDD patients and eight non-depressed controls. We found significantly lower maximal respiration rates, altered cytosolic basal calcium levels, and smaller soma size in NPCs derived from MDD patients. These findings are partially consistent with our earlier observations in MDD patient-derived fibroblasts. Furthermore, we differentiated MDD and control NPCs into iPS-neurons and analyzed their passive biophysical and active electrophysiological properties to investigate whether neuronal function can be related to altered mitochondrial activity and bioenergetics. Interestingly, MDD patient-derived iPS-neurons showed significantly lower membrane capacitance, a less hyperpolarized membrane potential, increased Na+ current density and increased spontaneous electrical activity. Our findings indicate that functional differences evident in fibroblasts derived from MDD patients are partially present after reprogramming to induced-NPCs, could relate to altered function of iPS-neurons and thus might be associated with the aetiology of major depressive disorder.

The cytokine IL-17A as a marker of treatment resistance in major depressive disorder?

Major depression is a complex disease and-among others, inflammation appears to play an important role in its pathophysiology. In this study, we investigated a broad range of cytokines in depressed patients. Plasma levels of interleukin (IL)-12/ IL-23p40, IL-15, IL-16, IL-17A, IL-1α, IL-7, tumor necrosis factorß and vascular endothelial growth factor were compared in 48 patients suffering from major depression before, after one and after six weeks of antidepressive treatment in relation to therapy response. Interestingly, the level of IL-17A turned out to rise significantly in the non-responder group compared to responder during antidepressive treatment. IL-17A is a pro-inflammatory cytokine that initiates the production of other cytokines, thereby inducing and mediating immune response. It is also involved in allergic and autoimmune-related diseases. The database investigating the role of IL-17A in major depressive disorder has grown within the last few years comparing levels of this cytokine in depressed patients versus healthy subjects. However, little is known about the expression of IL-17A during the course of antidepressive treatment. In summary, our study provides valuable evidence that this cytokine might serve as a marker of therapy resistance to antidepressants.

Prediction of Functional Consequences of Missense Mutations in ANO4 Gene.

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca2+-dependent ion channels and/or Ca2+-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.

BEST1 protein stability and degradation pathways differ between autosomal dominant Best disease and autosomal recessive bestrophinopathy accounting for the distinct retinal phenotypes.

Mutations in bestrophin-1 (BEST1) are associated with distinct retinopathies, notably three forms with autosomal dominant inheritance and one condition with an autosomal recessive mode of transmission. The molecular mechanisms underlying their distinct retinal phenotypes are mostly unknown. Although heterozygous missense mutations in BEST1 reveal dominant-negative effects in patients with autosomal dominant Best disease (BD), heterozygous mutations associated with autosomal recessive bestrophinopathy (ARB) display no disease phenotype. Here we show that the recessive mutations trigger a strong and fast protein degradation process in the endoplasmic reticulum (ER), thereby favoring a decreased stoichiometry of mutant versus normal BEST1 subunits in the assembly of the homo-pentameric BEST1 chloride channel. In contrast, dominant mutations escape ER-associated degradation and are subjected to a slightly delayed post-ER degradation via the endo-lysosomal degradation pathway. As a result, increased formation of a non-functional BEST1 channel occurs due to a roughly equimolar incorporation of normal and mutant BEST1 subunits into the channel complex. Taken together, our data provide insight into the molecular pathways of dominantly and recessively acting BEST1 missense mutations suggesting that the site of subcellular protein quality control as well as the rate and degree of mutant protein degradation are ultimately responsible for the distinct retinal disease phenotypes in BD and ARB.

Association of Chemokine (C-C Motif) Receptor 5 and Ligand 5 with Recovery from Major Depressive Disorder and Related Neurocognitive Impairment.

INTRODUCTION: Inflammatory processes play an important role in the pathophysiology of major depressive disorder (MDD), but their relevance for specific symptoms such as neurocognitive impairment is rarely investigated. METHODS: In this observational study, we investigated the changes of leukocyte chemokine (C-C motif) receptor 5 (CCR5) and ligand 5 (CCL5) mRNA levels and inflammatory cytokines in 60 MDD patients before (PRE) and after 5 weeks (W5) of antidepressive treatment in relation to therapy response and alterations in cognitive functions by means of the Cambridge Neuropsychological Test Automated Battery (CANTAB). We hypothesized that elevated CCR5 and CCL5 levels in depressed patients would decrease upon treatment and could differ with regard to cognitive impairment associated with MDD. RESULTS: Both CCR5 and CCL5 levels were significantly decreased in the responder group compared to nonresponders even before treatment. The cytokine IL-6 as a marker of inflammation in depression did not show a difference before treatment in future responders versus nonresponders, but decreased significantly upon antidepressive therapy. Regarding neurocognitive impairment in MDD patients, an increased misperception of the emotion "anger" after 5 weeks of treatment proved to be associated with a more pronounced change in CCR5, and the perception of the emotion "disgust" became faster along with a stronger decrease in CCL5 over the same time. Executive functions typically impaired in MDD patients were not markedly associated with alterations in CCR5/CCL5. DISCUSSION: CCR5 and CCL5 are important in the targeting of immune cells by HIV. This is the first study providing valuable hints that both CCR5 and CCL5 might also serve as markers of therapy response prediction in MDD. Regarding neurocognitive impairment in depression, CCR5 and CCL5 did not reveal characteristic changes upon MDD treatment such as executive functions, which are probably delayed. However, changes of emotional perception appear to be an earlier responding feature.

The Role of Chemokines in the Pathophysiology of Major Depressive Disorder.

Major depressive disorder (MDD) is a debilitating condition, whose high prevalence and multisymptomatic nature set its standing as a leading contributor to global disability. To better understand this psychiatric disease, various pathophysiological mechanisms have been proposed, including changes in monoaminergic neurotransmission, imbalance of excitatory and inhibitory signaling in the brain, hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis, and abnormalities in normal neurogenesis. While previous findings led to a deeper understanding of the disease, the pathogenesis of MDD has not yet been elucidated. Accumulating evidence has confirmed the association between chronic inflammation and MDD, which is manifested by increased levels of the C-reactive protein, as well as pro-inflammatory cytokines, such as Interleukin 1 beta, Interleukin 6, and the Tumor necrosis factor alpha. Furthermore, recent findings have implicated a related family of cytokines with chemotactic properties, known collectively as chemokines, in many neuroimmune processes relevant to psychiatric disorders. Chemokines are small (8-12 kDa) chemotactic cytokines, which are known to play roles in direct chemotaxis induction, leukocyte and macrophage migration, and inflammatory response propagation. The inflammatory chemokines possess the ability to induce migration of immune cells to the infection site, whereas their homeostatic chemokine counterparts are responsible for recruiting cells for their repair and maintenance. To further support the role of chemokines as central elements to healthy bodily function, recent studies suggest that these proteins demonstrate novel, brain-specific mechanisms including the modulation of neuroendocrine functions, chemotaxis, cell adhesion, and neuroinflammation. Elevated levels of chemokines in patient-derived serum have been detected in individuals diagnosed with major depressive disorder, bipolar disorder, and schizophrenia. Furthermore, despite the considerable heterogeneity of experimental samples and methodologies, existing biomarker studies have clearly demonstrated the important role of chemokines in the pathophysiology of psychiatric disorders. The purpose of this review is to summarize the data from contemporary experimental and clinical studies, and to evaluate available evidence for the role of chemokines in the central nervous system (CNS) under physiological and pathophysiological conditions. In light of recent results, chemokines could be considered as possible peripheral markers of psychiatric disorders, and/or targets for treating depressive disorders.

Microglial Pro-Inflammatory and Anti-Inflammatory Phenotypes Are Modulated by Translocator Protein Activation.

A key role of the mitochondrial Translocator Protein 18 KDa (TSPO) in neuroinflammation has been recently proposed. However, little is known about TSPO-activated pathways underlying the modulation of reactive microglia. In the present work, the TSPO activation was explored in an in vitro human primary microglia model (immortalized C20 cells) under inflammatory stimulus. Two different approaches were used with the aim to (i) pharmacologically amplify or (ii) silence, by the lentiviral short hairpin RNA, the TSPO physiological function. In the TSPO pharmacological stimulation model, the synthetic steroidogenic selective ligand XBD-173 attenuated the activation of microglia. Indeed, it reduces and increases the release of pro-inflammatory and anti-inflammatory cytokines, respectively. Such ligand-induced effects were abolished when C20 cells were treated with the steroidogenesis inhibitor aminoglutethimide. This suggests a role for neurosteroids in modulating the interleukin production. The highly steroidogenic ligand XBD-173 attenuated the neuroinflammatory response more effectively than the poorly steroidogenic ones, which suggests that the observed modulation on the cytokine release may be influenced by the levels of produced neurosteroids. In the TSPO silencing model, the reduction of TSPO caused a more inflamed phenotype with respect to scrambled cells. Similarly, during the inflammatory response, the TSPO silencing increased and reduced the release of pro-inflammatory and anti-inflammatory cytokines, respectively. In conclusion, the obtained results are in favor of a homeostatic role for TSPO in the context of dynamic balance between anti-inflammatory and pro-inflammatory mediators in the human microglia-mediated inflammatory response. Interestingly, our preliminary results propose that the TSPO expression could be stimulated by NF-κB during activation of the inflammatory response.

CRISPR-Cas9 Mediated TSPO Gene Knockout alters Respiration and Cellular Metabolism in Human Primary Microglia Cells.

The 18 kDa translocator protein (TSPO) is an evolutionary conserved cholesterol binding protein localized in the outer mitochondrial membrane. It has been implicated in the regulation of various cellular processes including oxidative stress, proliferation, apoptosis, and steroid hormone biosynthesis. Since the expression of TSPO in activated microglia is upregulated in various neuroinflammatory and neurodegenerative disorders, we set out to examine the role of TSPO in an immortalized human microglia C20 cell line. To this end, we performed a dual approach and used (i) lentiviral shRNA silencing to reduce TSPO expression, and (ii) the CRISPR/Cas9 technology to generate complete TSPO knockout microglia cell lines. Functional characterization of control and TSPO knockdown as well as knockout cells, revealed only low de novo steroidogenesis in C20 cells, which was not dependent on the level of TSPO expression or influenced by the treatment with TSPO-specific ligands. In contrast to TSPO knockdown C20 cells, which did not show altered mitochondrial function, the TSPO deficient knockout cells displayed a significantly decreased mitochondrial membrane potential and cytosolic Ca2+ levels, as well as reduced respiratory function. Performing the rescue experiment by lentiviral overexpression of TSPO in knockout cells, increased oxygen consumption and restored respiratory function. Our study provides further evidence for a significant role of TSPO in cellular and mitochondrial metabolism and demonstrates that different phenotypes of mitochondrial function are dependent on the level of TSPO expression.

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells.

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1(-/-)) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1(-/-) mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex--that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.

Macrophage-Derived Chemokine: A Putative Marker of Pharmacological Therapy Response in Major Depression?

INTRODUCTION: Inflammatory processes play an important and complex role in the pathophysiology of major depressive disorder (MDD), but, so far, no specific investigation of chemokines exists. METHODS: In this study, we investigated the changes of plasma chemokine levels (eotaxin-1, eotaxin-3, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1ß, and TARC) in 47 MDD patients before (PRE) and after 1 and 6 weeks of pharmacological treatment (POST1 and POST6) in relation to the response to antidepressive therapy. We hypothesized that the direction of alterations in levels of chemokines would significantly differ between the 2 groups, responders and nonresponders. RESULTS: Among the investigated chemokines, only the level of macrophage-derived chemokine (MDC) changed significantly in relation to therapy response. MDC levels were significantly elevated in the responder group at POST6. DISCUSSION: MDC is a constitutively expressed chemokine involved in the pathophysiology of infectious and neoplastic diseases. This is the first study providing valuable hints that MDC might serve as a marker of pharmacological therapy response in MDD.

Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance.

Bestrophin-1, an integral membrane protein encoded by the BEST1 gene, is localized predominantly to the basolateral membrane of the retinal pigment epithelium. Mutations in the BEST1 gene have been associated with Best vitelliforme macular dystrophy (BMD), a central retinopathy with autosomal dominant inheritance and variable penetrance. Over 120 disease-causing mutations are known, the majority of which result in amino acid substitutions within four mutational hot-spot regions in the highly conserved N-terminal half of the protein. Although initially thought to impair Cl⁻ channel function, the molecular pathology of BEST1 mutations is still controversial. We have analyzed the subcellular localization of 13 disease-associated BEST1 mutant proteins in polarized MDCK II cells, an established model of apical to basolateral protein sorting. Immunostaining demonstrated that nine of the 13 mutant proteins failed to integrate into the cell membrane. The defective proteins were predominantly retained in the cytoplasm, whereas wild-type bestrophin-1 revealed cell membrane localization. Functional analysis of I⁻ fluxes in HEK-293 cells showed that all mutants exhibited a significant reduction in anion conductance. Our data indicate that defective intracellular trafficking could be a common cause of BMD accompanied by impaired anion conductance, representing a loss of anion channel function that is probably due to mistargeting of mutant protein.

Impact of Translocator Protein 18 kDa (TSPO) Deficiency on Mitochondrial Function and the Inflammatory State of Human C20 Microglia Cells.

Microglia are the resident immune cells of the central nervous system. Upon stimulus presentation, microglia polarize from a resting to an activated state. Microglial translocator protein 18 kDa (TSPO) is considered a marker of inflammation. Here, we characterized the role of TSPO by investigating the impact of TSPO deficiency on human microglia. We used TSPO knockout (TSPO-/-) variants of the human C20 microglia cell line. We found a significant reduction in the TSPO-associated protein VDAC1 in TSPO-/- cells compared to control cells. Moreover, we assessed the impact of TSPO deficiency on calcium levels and the mitochondrial membrane potential. Cytosolic and mitochondrial calcium concentrations were increased in TSPO-/- cell lines, whereas the mitochondrial membrane potential tended to be lower. Assessment of the mitochondrial DNA copy number via RT-PCR revealed a decreased amount of mtDNA in the TSPO-/- cells when compared to controls. Moreover, the metabolic profiles of C20 cells were strongly dependent on the glycolytic pathway. However, TSPO depletion did not affect the cellular metabolic profile. Measurement of the mRNA expression levels of the pro-inflammatory mediators revealed an attenuated response to pro-inflammatory stimuli in TSPO-depleted cells, implying a role for the TSPO protein in the process of microglial polarization.

Translocator protein (18kDA) (TSPO) marks mesenchymal glioblastoma cell populations characterized by elevated numbers of tumor-associated macrophages.

TSPO is a promising novel tracer target for positron-emission tomography (PET) imaging of brain tumors. However, due to the heterogeneity of cell populations that contribute to the TSPO-PET signal, imaging interpretation may be challenging. We therefore evaluated TSPO enrichment/expression in connection with its underlying histopathological and molecular features in gliomas. We analyzed TSPO expression and its regulatory mechanisms in large in silico datasets and by performing direct bisulfite sequencing of the TSPO promotor. In glioblastoma tissue samples of our TSPO-PET imaging study cohort, we dissected the association of TSPO tracer enrichment and protein labeling with the expression of cell lineage markers by immunohistochemistry and fluorescence multiplex stains. Furthermore, we identified relevant TSPO-associated signaling pathways by RNA sequencing.We found that TSPO expression is associated with prognostically unfavorable glioma phenotypes and that TSPO promotor hypermethylation is linked to IDH mutation. Careful histological analysis revealed that TSPO immunohistochemistry correlates with the TSPO-PET signal and that TSPO is expressed by diverse cell populations. While tumor core areas are the major contributor to the overall TSPO signal, TSPO signals in the tumor rim are mainly driven by CD68-positive microglia/macrophages. Molecularly, high TSPO expression marks prognostically unfavorable glioblastoma cell subpopulations characterized by an enrichment of mesenchymal gene sets and higher amounts of tumor-associated macrophages.In conclusion, our study improves the understanding of TSPO as an imaging marker in gliomas by unveiling IDH-dependent differences in TSPO expression/regulation, regional heterogeneity of the TSPO PET signal and functional implications of TSPO in terms of tumor immune cell interactions.

TSPO acts as an immune resistance gene involved in the T cell mediated immune control of glioblastoma.

Glioblastoma (GB) IDH-wildtype is the most malignant primary brain tumor. It is particularly resistant to current immunotherapies. Translocator protein 18 kDa (TSPO) is upregulated in GB and correlates with malignancy and poor prognosis, but also with increased immune infiltration. Here, we studied the role of TSPO in the regulation of immune resistance of human GB cells. The role of TSPO in tumor immune resistance was experimentally determined in primary brain tumor initiating cells (BTICs) and cell lines through genetic manipulation of TSPO expression and subsequent cocultures with antigen specific cytotoxic T cells and autologous tumor-infiltrating T cells. Death inducing intrinsic and extrinsic apoptotic pathways affected by TSPO were investigated. TSPO-regulated genes mediating apoptosis resistance in BTICs were identified through gene expression analysis and subsequent functional analyses. TSPO transcription in primary GB cells correlated with CD8+ T cell infiltration, cytotoxic activity of T cell infiltrate, expression of TNFR and IFNGR and with the activity of their downstream signalling pathways, as well as with the expression of TRAIL receptors. Coculture of BTICs with tumor reactive cytotoxic T cells or with T cell-derived factors induced TSPO up-regulation through T cell derived TNFα and IFNγ. Silencing of TSPO sensitized BTICs against T cell-mediated cytotoxicity. TSPO selectively protected BTICs against TRAIL-induced apoptosis by regulating apoptosis pathways. TSPO also regulated the expression of multiple genes associated with resistance against apoptosis. We conclude that TSPO expression in GB is induced through T cell-derived cytokines TNFα and IFNγ and that TSPO expression protects GB cells against cytotoxic T cell attack through TRAIL. Our data thereby provide an indication that therapeutic targeting of TSPO may be a suitable approach to sensitize GB to immune cell-mediated cytotoxicity by circumventing tumor intrinsic TRAIL resistance.

Differential effects of the translocator protein 18 kDa (TSPO) ligand etifoxine and the benzodiazepine alprazolam on startle response to predictable threat in a NPU-threat task after acute and short-term treatment.

RATIONALE: Benzodiazepines have been extensively investigated in experimental settings especially after single administration, which mostly revealed effects on unpredictable threat (U-threat) rather than predictable threat (P-threat). Given the need for pharmacological alternatives with a preferable side-effect profile and to better represent clinical conditions, research should cover also other anxiolytics and longer application times. OBJECTIVES: The present study compared the acute and short-term effects of the translocator protein 18 kDa (TSPO) ligand etifoxine and the benzodiazepine alprazolam on P-threat and U-threat while controlling for sedation. METHODS: Sixty healthy male volunteers, aged between 18 and 55 years, were randomly assigned to receive a daily dose of either 150 mg etifoxine, 1.5 mg alprazolam, or placebo for 5 days. On days 1 and 5 of intake, they performed a NPU-threat task including neutral (N), predictable (P), and unpredictable (U) conditions, while startle responsivity and self-reports were studied. Sedative effects were assessed using a continuous performance test. RESULTS: Neither alprazolam nor etifoxine affected startle responsivity to U-threat on any of the testing days. While etifoxine reduced the startle response to P-threat on day 1 of treatment for transformed data, a contrary effect of alprazolam was found for raw values. No effects on self-reports and no evidence of sedation could be observed for either drug. CONCLUSIONS: None of the anxiolytic substances had an impact on startle potentiation to U-threat even after several days of intake. The effects of the anxiolytics on startle responsivity to P-threat as well as implications for future studies are discussed.

Potential Involvement of Extracellular Citrate in Brain Tumor Progression.

Brain tissue is known to have elevated citrate levels, necessary to regulate ion chelation, neuron excitability, and are also necessary for the supply of necessary energy substrates to neurons. Importantly, citrate also acts as a central substrate in cancer metabolism. Recent studies have shown that extracellular citrate levels in the brain undergo significant changes during tumor development and may play a dual role in tumor progression, as well as cancer cell aggressiveness. In the present article, we review available literature describing changes of citrate levels in brain tissue, blood, and cerebrospinal fluid, as well as intracellular alterations during tumor development before and after metastatic progression. Based on the available literature and our recent findings, we hypothesize that changes in extracellular citrate levels may be related to the increased consumption of this metabolite by cancer cells. Interestingly, cancerassociated cells, including reactive astrocytes, might be a source of citrate. Extracellular citrate uptake mechanisms, as well as potential citrate synthesis and release by surrounding stroma, could provide novel targets for anti-cancer treatments of primary brain tumors and brain metastases.

Dissociation of endocrine responses to the Trier Social Stress Test in Virtual Reality (VR-TSST) by the benzodiazepine alprazolam and the translocator protein 18 kDa (TSPO) ligand etifoxine.

BACKGROUND: Activity of the two major stress systems, the hypothalamic-pituitary-adrenal (HPA) and the sympathetic-adrenal-medullary (SAM) axis, has already been shown to be modulated by different compounds that bind to the central benzodiazepine receptor. Less is known about ligands that modulate the peripheral benzodiazepine receptor - meanwhile known as the translocator protein 18 kDa (TSPO) - which constitute promising candidates in the search of novel anxiolytics. To close this gap, the present study compared the effects of the benzodiazepine alprazolam and the TSPO ligand etifoxine on responses of the HPA and SAM axes to the Trier Social Stress Test, a standardized paradigm to induce acute psychosocial stress in humans, performed in Virtual Reality (VR-TSST). METHODS: Sixty healthy males, aged between 18 and 55 years, were randomly assigned to receive either a daily dose of 1.5 mg alprazolam, 150 mg etifoxine, or placebo over five days. On the last day of intake, they were exposed to the VR-TSST. We assessed changes of salivary cortisol, allopregnanolone, (nor-) epinephrine in serum, TSPO expression in platelets as well as heart rate (HR), skin conductance level (SCL) and self-reports in response to the stress task. Repeated measures ANOVAs were conducted to examine treatment effects on these stress response variables during the course of VR-TSST. RESULTS: The response of salivary cortisol to the VR-TSST was significantly blunted in participants pre-treated with alprazolam but was not affected by etifoxine. While levels of allopregnanolone, epinephrine and norepinephrine increased in response to stress, TSPO expression decreased. None of those endocrine stress markers was affected by the active treatments, whereas TSPO expression increased after etifoxine administration over all study days. There were no effects of the two anxiolytics on HR, SCL or any self-report measurement. CONCLUSION: The current study confirmed the attenuating effects of benzodiazepines on stress-induced HPA axis activity but did not reveal a comparable effect of the TSPO ligand etifoxine. The long-term consequences of a pharmacologically blunted response of the HPA axis to an acute stressor should be further elucidated. Due to the missing effects of etifoxine on stress-related parameters in our sample of healthy subjects, it might be concluded that the therapeutic effects of this TSPO ligand are restricted to stronger or pathological stress responses, respectively.