Several evidence indicate that metabolic alterations play a pivotal role in cancer development. Here, we report that the mitochondrial uncoupling protein 2 (UCP2) sustains the metabolic shift from mitochondrial oxidative phosphorylation (mtOXPHOS) to glycolysis in pancreas cancer cells. Indeed, we show that UCP2 sensitizes pancreas cancer cells to the treatment with the glycolytic inhibitor 2-deoxy-D-glucose. Through a bidimensional electrophoresis analysis, we identify 19 protein species differentially expressed after treatment with the UCP2 inhibitor genipin and, by bioinformatic analyses, we show that these proteins are mainly involved in metabolic processes. In particular, we demonstrate that the antioxidant UCP2 induces the expression of hnRNPA2/B1, which is involved in the regulation of both GLUT1 and PKM2 mRNAs, and of lactate dehydrogenase (LDH) increasing the secretion of L-lactic acid. We further demonstrate that the radical scavenger N-acetyl-L-cysteine reverts hnRNPA2/B1 and PKM2 inhibition by genipin indicating a role for reactive oxygen species in the metabolic reprogramming of cancer cells mediated by UCP2. We also observe an UCP2-dependent decrease in mtOXPHOS complex I (NADH dehydrogenase), complex IV (cytochrome c oxidase), complex V (ATPase) and in mitochondrial oxygen consumption, suggesting a role for UCP2 in the counteraction of pancreatic cancer cellular respiration. All these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation with the concomitant metabolic shift from mtOXPHOS to the glycolytic pathway.

Proper mitochondrial function is of great importance to maintain efficient cardiac function. Dysregulated fatty acid oxidation (FAO) or glucose metabolism is an underlying cause of cardiovascular diseases and metabolic syndrome. The mitochondrial uncoupling protein 3 (UCP3) is proposed to have a protective role in heart disease. However, the physiological function of UCP3 is under debate, and its role as an anti-oxidative protein is critically discussed. UCP3 expression was shown to coincide with FAO, which strengthens the hypothesis about UCP3 involvement in metabolism.

Reprogrammed cellular energy metabolism is considered one of the cancer hallmarks. Most cells use oxidative phosphorylation under physiological conditions, whereas malignant cells switch to aerobic glycolysis. Uncontrolled cell growth and proliferation lead to glucose shortage, which cancer cells may bypass by glutaminolysis. Previous studies have shown that mouse neuroblastoma cells (N18TG2) upregulate mitochondrial uncoupling protein 2 (UCP2) in the presence of glutamine under glucose deficiency [1].

The cyclin-dependent kinase 6 (CDK6) regulates the transition through the G1-phase of the cell cycle, but also acts as a transcriptional regulator. As such CDK6 regulates cell survival or cytokine secretion together with STATs, AP-1 or NF-κB. In the hematopoietic system, CDK6 regulates T cell development and promotes leukemia and lymphoma. CDK4/6 kinase inhibitors are FDA approved for treatment of breast cancer patients and have been reported to enhance T cell-mediated anti-tumor immunity. The involvement of CDK6 in T cell functions remains enigmatic. We here investigated the role of CDK6 in CD8+ T cells, using previously generated CDK6 knockout (Cdk6-/-) and kinase-dead mutant CDK6 (Cdk6K43M) knock-in mice. RNA-seq analysis indicated a role of CDK6 in T cell metabolism and interferon (IFN) signaling. To investigate whether these CDK6 functions are T cell-intrinsic, we generated a T cell-specific CDK6 knockout mouse model (Cdk6fl/fl CD4-Cre). T cell-intrinsic loss of CDK6 enhanced mitochondrial respiration in CD8+ T cells, but did not impact on cytotoxicity and production of the effector cytokines IFN-γ and TNF-α by CD8+ T cells in vitro. Loss of CDK6 in peripheral T cells did not affect tumor surveillance of MC38 tumors in vivo. Similarly, while we observed an impaired induction of early responses to type I IFN in CDK6-deficient CD8+ T cells, we failed to observe any differences in the response to LCMV infection upon T cell-intrinsic loss of CDK6 in vivo. This apparent contradiction might at least partially be explained by the reduced expression of Socs1, a negative regulator of IFN signaling, in CDK6-deficient CD8+ T cells. Therefore, our data are in line with a dual role of CDK6 in IFN signaling; while CDK6 promotes early IFN responses, it is also involved in the induction of a negative feedback loop. These data assign CDK6 a role in the fine-tuning of cytokine responses.

The neuropeptide galanin (GAL), which is expressed in limbic brain structures, has a strong impact on the regulation of mood and behavior. GAL exerts its effects via three G protein-coupled receptors (GAL1–3-R). Little is known about the effects of aging and loss of GAL-Rs on hippocampal-mediated processes connected to neurogenesis, such as learning, memory recall and anxiety, and cell proliferation and survival in the dorsal dentate gyrus (dDG) in mice. Our results demonstrate that loss of GAL3-R, but not GAL2-R, slowed learning and induced anxiety in older (12–14-month-old) mice. Lack of GAL2-R increased cell survival (BrdU incorporation) in the dDG of young mice. However, normal neurogenesis was observed in vitro using neural stem and precursor cells obtained from GAL2-R and GAL3-R knockouts upon GAL treatment. Interestingly, we found sub-strain differences between C57BL/6J and C57BL/6N mice, the latter showing faster learning, less anxiety and lower cell survival in the dDG. We conclude that GAL-R signaling is involved in cognitive functions and can modulate the survival of cells in the neurogenic niche, which might lead to new therapeutic applications. Furthermore, we observed that the mouse sub-strain had a profound impact on the behavioral parameters analyzed and should therefore be carefully considered in future studies.

Vertebrate respiratory chain complex III consists of eleven subunits. Mutations in five subunits either mitochondrial (MT-CYB) or nuclear (CYC1, UQCRC2, UQCRB, and UQCRQ) encoded have been reported. Defects in five further factors for assembly (TTC19, UQCC2, and UQCC3) or iron-sulphur cluster loading (BCS1L and LYRM7) cause complex III deficiency. Here, we report a second patient with UQCC2 deficiency. This girl was born prematurely; pregnancy was complicated by intrauterine growth retardation and oligohydramnios. She presented with respiratory distress syndrome, developed epileptic seizures progressing to status epilepticus, and died at day 33. She had profound lactic acidosis and elevated urinary pyruvate. Exome sequencing revealed two homozygous missense variants in UQCC2, leading to a severe reduction of UQCC2 protein. Deficiency of complexes I and III was found enzymatically and on the protein level. A review of the literature on genetically distinct complex III defects revealed that, except TTC19 deficiency, the biochemical pattern was very often a combined respiratory chain deficiency. Besides complex III, typically, complex I was decreased, in some cases complex IV. In accordance with previous observations, the presence of assembled complex III is required for the stability or assembly of complexes I and IV, which might be related to respirasome/supercomplex formation.

The ketogenic diet (KD), a high-fat low-carbohydrate diet, has shown some efficacy in the treatment of certain types of tumors such as brain tumors and neuroblastoma. These tumors are characterized by the Warburg effect. Because renal cell carcinoma (RCC) presents similar energetic features as neuroblastoma, KD might also be effective in the treatment of RCC. To test this, we established xenografts with RCC 786-O cells in CD-1 nu/nu mice and then randomized them to a control diet or to KDs with different triglyceride contents. Although the KDs tended to reduce tumor growth, mouse survival was dramatically reduced due to massive weight loss. A possible explanation comes from observations of human RCC patients, who often experience secondary non-metastatic hepatic dysfunction due to secretion of high levels of inflammatory cytokines by the RCCs. Measurement of the mRNA levels of tumor necrosis factor alpha (TNFα) and interleukin-6 revealed high expression in the RCC xenografts compared to the original 786-O cells. The expression of TNFα, interleukin-6 and C-reactive protein were all increased in the livers of tumor-bearing mice, and KD significantly boosted their expression. KDs did not cause weight loss or liver inflammation in healthy mice, suggesting that KDs are per se safe, but might be contraindicated in the treatment of RCC patients presenting with Stauffer's syndrome, because they potentially worsen the associated hepatic dysfunction.

Neuroblastoma (NB) is a pediatric malignancy characterized by a marked reduction in aerobic energy metabolism. Recent preclinical data indicate that targeting this metabolic phenotype by a ketogenic diet (KD), especially in combination with calorie restriction, slows tumor growth and enhances metronomic cyclophosphamide (CP) therapy of NB xenografts. Because calorie restriction would be contraindicated in most cancer patients, the aim of the present study was to optimize the KD such that the tumors are sensitized to CP without the need of calorie restriction. In a NB xenograft model, metronomic CP was combined with KDs of different triglyceride compositions and fed to CD1-nu mice ad libitum. Metronomic CP in combination with a KD containing 8-carbon medium-chain triglycerides exerted a robust anti-tumor effect, suppressing growth and causing a significant reduction of tumor blood-vessel density and intratumoral hemorrhage, accompanied by activation of AMP-activated protein kinase in NB cells. Furthermore, the KDs caused a significant reduction in the serum levels of essential amino acids, but increased those of serine, glutamine and glycine. Our data suggest that targeting energy metabolism by a modified KD may be considered as part of a multimodal treatment regimen to improve the efficacy of classic anti-NB therapy.

One of the causes of preeclampsia (PE) is due to an imbalance of inflammatory and anti-inflammatory proteins, as well as hyperactivity of the sympathetic nervous system. Previous studies have already revealed the involvement of the neuropeptides galanin (GAL) and oxytocin (OXT) in inflammatory processes and their modulatory role with respect to the autonomous system 1, 2. Despite those findings, evidence for a relation of these hormones in the pathogenesis of PE is missing so far. Aim of the study is to characterise the above mentioned peptide systems regarding their expression patterns in the placenta, umbilical cord and plasma of PE patients and healthy controls. Placental (maternal and fetal side) and umbilical cord tissues have been collected during caesarean section from control and PE patients. Furthermore blood samples have been taken at the day of the surgery and four days postpartum. Biopsies have been analysed for the expression of GAL, GAL1,3- and OXT-receptors (GAL1, GAL3, OXTR). Additionally, differentiated analysis of maternal and fetal tissue was performed to reveal potential different expression patterns. Preliminary results of the IHC revealed a clear expression of GAL3 in trophoblasts of PE placentas, whereas controls showed an increase expression of GAL3 on endothelial cells. Moreover an increased expression of GAL1 and GAL on endothelial cells has been detected in controls compared to PE. Different patterns were also visible regarding the OXTR with higher expression levels in controls compared to PE. Furthermore, PE placentas showed local expression differences (fetal > maternal side), which was not apparent in controls. The different expression patterns of the GAL-system and the OXTR still have to be verified by Western Blot analyses, qPCR and IHC. However, their preliminary detection in placental tissue could be a first hint regarding the involvement of those systems in the inflammatory processes of PE.

Glucagon-like peptide-1 (GLP-1) receptor agonists are indicated for the treatment of Type 2 diabetes and obesity, but can cause nausea and emesis in some patients. GLP-1 receptors are distributed widely in the brain, where they contribute to mechanisms of emesis, reduced appetite and aversion, but it is not known if these centrally located receptors also contribute to a modulation of gastric slow wave activity, which is linked causally to nausea. Our aim was to investigate the potential of the GLP-1 receptor agonist, exendin-4, administered into the 3rd ventricle to modulate emesis, feeding and gastric slow wave activity. Thermoregulation and cardiovascular parameters were also monitored, as they are disturbed during nausea.Ferrets were used as common laboratory rodents do not have an emetic reflex. A guide cannula was implanted into the 3rd ventricle for delivering a previously established dose of exendin-4 (10 nmol), which had been shown to induce emesis and behaviours indicative of 'nausea'. Radiotelemetry recorded gastric myoelectric activity (GMA; slow waves), blood pressure and heart rate variability (HRV), and core temperature; food intake and behaviour were also assessed.Exendin-4 (10 nmol, i.c.v.) decreased the dominant frequency of GMA, with an associated increase in the percentage of bradygastric power (lasting ~ 4 h). Food intake was inhibited in all animals, with 63% exhibiting emesis. Exendin-4 also increased blood pressure (lasting ~ 24 h) and heart rate (lasting ~ 7 h), decreased HRV (lasting ~ 24 h), and caused transient hyperthermia. None of the above parameters were emesis-dependent.The present study shows for the first time that gastric slow waves may be modulated by GLP-1 receptors in the brain through mechanisms that appear independent from emesis. Taken together with a reduction in HRV, the findings are consistent with changes associated with the occurrence of nausea in humans.

Galanin (GAL) has been shown to be a vasoactive (neuro)-peptide which plays a role in a range of inflammatory diseases. The effects of GAL are mediated via three G protein-coupled receptors (GAL1-3). The aim of the present study was to elucidate which galanin receptor is expressed by endothelial cells and which function this receptor plays in the progression of psoriasis in vitro and in vivo. Healthy and psoriatric human skin was analyzed for expression of GAL1-3 by immunohistochemistry. Psoriasis-like skin inflammation was induced via daily topical application of 5% imiquimod (IMQ) (Aldara) on the shaved and depilated back skin of 8-10 week old male and female GAL2-knockout (KO) and GAL3-KO mice for 6 consecutive days. In healthy human skin GAL3 staining was observed around large, mature vessels. In psoriatric skin GAL3 staining was also observed in large, mature vessels. In addition, in newly formed micro-vessels GAL3 staining co-localized with nestin, a marker of neovascularization. Psoriasis development in skin of GAL3-KO animals resulted in a significant lower psoriasis disease severity score, mRNA levels of IL-17A, IL-22, IL-23 and TNF-α and reduced density of CD31 positive vessels compared to wild types on day 4. Neutrophil derived myeloperoxidase activity was significantly lowered in GAL3-KO mice compared to WT mice which was in line with reduced neutrophil infiltration in psoriatric skin of GAL3-KO mice. The total number of macrophages and mast cells in the skin was not affected by lack of GAL3. GAL2-KO mice did not differ significantly from WT mice at both the macroscopic and molecular levels in their inflammatory response to IMQ treatment. Our data indicate that GAL3, but not GAL2, plays an important role in neovascularization and psoriasis-like skin inflammation.

Previously, we have shown that the neuropeptide galanin inhibits neurogenic inflammation and associated plasma extravasation in the murine skin via activation of the galanin receptor 3 (GAL3). The aim of the present study was to elucidate if C57/BL6N mice lacking GAL3 (GAL3-KO) display an altered course of psoriasis. Via daily topical application of 5% imiquimod (Aldara) on the shaved back skin of mice for 6 consecutive days psoriasis-like skin inflammation was induced. The analysis focused on psoriasis disease severity, epidermal thickening, changes in vascularization, macrophage and neutrophil infiltration and mRNA levels of specific cytokines. To address the role of GAL3 on vessel formation we used the human microvascular endothelial cell line HMEC-1 and blocked GAL3 signaling via a specific antagonist namely SNAP 37889 and analysed the changes in HMEC-1 typical tube formation. Psoriasis development was altered in GAL3-KO animals resulting in a significant lower psoriasis disease severity score and significantly reduced mRNA levels of IL-12b, IL-17A, IL-22 and IL-23 at day 4. We also observed significantly reduced density of CD31 positive vessels in the skin of GAL3-KO mice compared to wild types at day 4 indicating delayed neovascularisation. Blocking GAL3in vitro for 2 h with 10 μM SNAP 37889 in HMEC-1 cells revealed a significant reduction of tube formation. Interestingly, we detected no changes between genotypes in the expression of vascularisation-related genes like VEGF, Angiopoietin I and II. The total number of F4/80 positive macrophages in the skin sections as well as the neutrophil derived myeloperoxidase activity was not affected by lack of GAL3. Our data indicate that GAL3 plays an important role in psoriasis-like skin inflammation and neovascularisation.

Neurogenic inflammation mediated by peptidergic sensory nerves has a crucial impact on the pathogenesis of various joint diseases. Galanin is a regulatory sensory neuropeptide, which has been shown to attenuate neurogenic inflammation, modulate neutrophil activation, and be involved in the development of adjuvant arthritis, but our current understanding about its targets and physiological importance is incomplete. Among the receptors of galanin (GAL1-3), GAL3 has been found to be the most abundantly expressed in the vasculature and on the surface of some immune cells. However, since there are minimal in vivo data on the role of GAL3 in joint diseases, we analyzed its involvement in different inflammatory mechanisms of the K/BxN serum transfer-model of autoimmune arthritis employing GAL 3 gene-deficient mice. After arthritis induction, GAL3 knockouts demonstrated increased clinical disease severity and earlier hindlimb edema than wild types. Vascular hyperpermeability determined by in vivo fluorescence imaging was also elevated compared to the wild-type controls. However, neutrophil accumulation detected by in vivo luminescence imaging or arthritic mechanical hyperalgesia was not altered by the lack of the GAL3 receptor. Our findings suggest that GAL3 has anti-inflammatory properties in joints by inhibiting vascular hyperpermeability and consequent edema formation.

SpringerPlus 2015, 4(Suppl 1):L1 MicroRNAs (miRNAs) are short, 22-25 nucleotide long transcripts that may suppress entire signaling pathways by interacting with the 3'-untranslated region (3'-UTR) of coding mRNA targets, interrupting translation and inducing degradation of these targets.The long 3'-UTRs of brain transcripts compared to other tissues predict important roles for brain miRNAs.Supporting this notion, we found that brain miRNAs co-evolved with their target transcripts, that non-coding pseudogenes with miRNA recognition elements compete with brain coding mRNAs on their miRNA interactions, and that Single Nucleotide Polymorphisms (SNPs) on such pseudogenes are enriched in mental diseases including autism and schizophrenia, but not Alzheimer's disease (AD).Focusing on evolutionarily conserved and primate-specifi c miRNA controllers of cholinergic signaling ('CholinomiRs'), we fi nd modifi ed CholinomiR levels in the brain and/or nucleated blood cells of patients with AD and Parkinson's disease, with treatment-related diff erences in their levels and prominent impact on the cognitive and anti-infl ammatory consequences of cholinergic signals.Examples include the acetylcholinesterase (AChE)-targeted evolutionarily conserved miR-132, whose levels decline drastically in the AD brain.Furthermore, we found that interruption of AChE mRNA's interaction with the primatespecifi c CholinomiR-608 in carriers of a SNP in the AChE's miR-608 binding site induces domino-like eff ects that reduce the levels of many other miR-608 targets.Young, healthy carriers of this SNP express 40% higher brain AChE activity than others, potentially aff ecting the responsiveness to AD's anti-AChE therapeutics, and show elevated trait anxiety, infl ammation and hypertension.Non-coding regions aff ecting miRNA-target interactions in neurodegenerative brains thus merit special attention.

AIMS:Polymorphonuclear neutrophils are key players in innate immunity. The innate immune system needs to be tightly controlled to ensure proper activation but also no overactivation. Galanin has been shown to regulate inflammatory reactions, and therefore, we aimed to elucidate the expression of galanin and its three receptors (GAL1 -GAL3 ) in polymorphonuclear neutrophils and to evaluate whether galanin exerts direct or indirect effects on human and murine polymorphonuclear neutrophils.METHODS:Human peripheral polymorphonuclear neutrophils were isolated from fresh blood of healthy donors, and murine polymorphonuclear neutrophils were isolated from bone marrow of C57BL/6N mice. Gene expression was evaluated by qRT-PCR. As a marker for polymorphonuclear neutrophil activation, CD11b integrin surface expression was measured by FACS analysis. Furthermore, a label-free technology measuring ligand-induced dynamic mass redistribution was used to evaluate the response of polymorphonuclear neutrophils to galanin.RESULTS:GAL2 receptor expression was found in both human and murine polymorphonuclear neutrophils, galanin and GAL3 receptor were exclusively expressed in murine bone marrow polymorphonuclear neutrophils, and GAL1 receptor was not detectable in polymorphonuclear neutrophils of either species. Galanin treatment was not able to induce CD11b integrin surface expression or dynamic mass redistribution in human polymorphonuclear neutrophils and murine bone marrow polymorphonuclear neutrophils. However, galanin treatment significantly enhanced the response of polymorphonuclear neutrophils of both species to interleukin-8.CONCLUSION:Galanin can be regarded as an immunomodulatory peptide as it can sensitize polymorphonuclear neutrophils towards pro-inflammatory cytokines in humans and mice.

Galanin and its receptors (GAL1, GAL2, GAL3) modulate a range of neuronal, immune and vascular activities. In vivo administration of SNAP 37889 (1-phenyl-3-[[3-(trifluoromethyl)phenyl]imino]-1H-indol-2-one), a potent small non-peptidergic antagonist of GAL3, was reported to reduce anxiety- and depression-related behavior, ethanol consumption, and antagonizes the effect of galanin on plasma extravasation in rodent models. Accordingly, SNAP 37889 has been proposed as a potential therapeutic agent to treat anxiety and depression disorders. Therefore, we evaluated the toxicity of SNAP 37889 to different cell types. Our experiments revealed that SNAP 37889 (≥10μM) induced apoptosis in epithelial (HMCB) and microglial (BV-2) cell lines expressing endogenous GAL3, in peripheral blood mononuclear cells and promyelocytic leukemia cells (HL-60) expressing GAL2, and in a neuronal cell line (SH-SY5Y) lacking galanin receptor expression altogether. In conclusion, SNAP 37889 is toxic to a variety of cell types independent of GAL3 expression. We caution that the clinical use of SNAP 37889 at doses that might be used to treat anxiety- or depression- related diseases could have unexpected non-galanin receptor-mediated toxicity, especially on immune cells.