Negative Feedback Signal Research Articles

Complementary synaptic distribution of enzymes responsible for synthesis and inactivation of the endocannabinoid 2-arachidonoylglycerol in the human hippocampus

Clinical and experimental evidence demonstrates that endocannabinoids play either beneficial or adverse roles in many neurological and psychiatric disorders. Their medical significance may be best explained by the emerging concept that endocannabinoids are essential modulators of synaptic transmission throughout the central nervous system. However, the precise molecular architecture of the endocannabinoid signaling machinery in the human brain remains elusive. To address this issue, we investigated the synaptic distribution of metabolic enzymes for the most abundant endocannabinoid molecule, 2-arachidonoylglycerol (2-AG), in the postmortem human hippocampus. Immunostaining for diacylglycerol lipase-α (DGL-α), the main synthesizing enzyme of 2-AG, resulted in a laminar pattern corresponding to the termination zones of glutamatergic pathways. The highest density of DGL-α-immunostaining was observed in strata radiatum and oriens of the cornu ammonis and in the inner third of stratum moleculare of the dentate gyrus. At higher magnification, DGL-α-immunopositive puncta were distributed throughout the neuropil outlining the immunonegative main dendrites of pyramidal and granule cells. Electron microscopic analysis revealed that this pattern was due to the accumulation of DGL-α in dendritic spine heads. Similar DGL-α-immunostaining pattern was also found in hippocampi of wild-type, but not of DGL-α knockout mice. Using two independent antibodies developed against monoacylglycerol lipase (MGL), the predominant enzyme inactivating 2-AG, immunostaining also revealed a laminar and punctate staining pattern. However, as observed previously in rodent hippocampus, MGL was enriched in axon terminals instead of postsynaptic structures at the ultrastructural level. Taken together, these findings demonstrate the post- and presynaptic segregation of primary enzymes responsible for synthesis and elimination of 2-AG, respectively, in the human hippocampus. Thus, molecular architecture of the endocannabinoid signaling machinery supports retrograde regulation of synaptic activity, and its similar blueprint in rodents and humans further indicates that 2-AG's physiological role as a negative feed-back signal is an evolutionarily conserved feature of excitatory synapses.

Lasker Lauds Leptin

This year, the Albert Lasker Basic Medical Research Award will be shared by Douglas Coleman and Jeffrey Friedman for their discovery of leptin, a hormone that regulates appetite and body weight. By uncovering a critical physiologic system, their discovery markedly accelerated our capacity to apply molecular and genetic techniques to understand obesity.

The Molecularity of the Newman−Kwart Rearrangement

It was recently reported that the venerable Newman-Kwart rearrangement (1→2) proceeds via mixed first- and second-order kinetics. Prior to this, the rearrangement had been considered to proceed exclusively via an intramolecular O(Ar)→S(Ar) migration. A new bimolecular pathway, possibly involving an 8-membered cyclic transition state, was proposed to account for reaction rates that increased disproportionately with substrate concentration under microwave heating conditions. We report a reanalysis of the kinetics and molecularity of the rearrangement of N,N-dimethyl O-(p-nitrophenyl)thiocarbamate 1a in N,N-dimethylacetamide solvent. Using HPLC, isotopic labeling ((2)H, (18)O, (34)S), and ESI-ICRMS methods, we show that there is no evidence for a bimolecular pathway en route to 2a, with near-perfect exponential decay in 1a at concentrations ranging from 0.11 to 4.70 M. Instead, it is demonstrated that under the microwave heating conditions, a delayed negative feedback signal to the microwave power balancing loop results in oscillatory reaction overheating. Due to higher tan δ in the solute, the amplitude of this oscillation increases with the concentration of 1a, and this phenomenon best accounts for the kinetic behavior previously misinterpreted as being mixed first- and second-order in nature.

Progesterone Treatment Inhibits and Dihydrotestosterone (DHT) Treatment Potentiates Voltage-Gated Calcium Currents in Gonadotropin-Releasing Hormone (GnRH) Neurons

GnRH neurons are central regulators of fertility, and their activity is modulated by steroid feedback. In normal females, GnRH secretion is regulated by estradiol and progesterone (P). Excess androgens present in hyperandrogenemic fertility disorders may disrupt communication of negative feedback signals from P and/or independently stimulate GnRH release. Voltage-gated calcium channels (VGCCs) are important in regulating excitability and hormone release. Estradiol alters VGCCs in a time-of-day-dependent manner. To further elucidate ovarian steroid modulation of GnRH neuron VGCCs, we studied the effects of dihydrotestosterone (DHT) and P. Adult mice were ovariectomized (OVX) or OVX and treated with implants containing DHT (OVXD), estradiol (OVXE), estradiol and DHT (OVXED), estradiol and P (OVXEP), or estradiol, DHT, and P (OVXEDP). Macroscopic calcium current (I(Ca)) was recorded in the morning or afternoon 8-12 d after surgery using whole-cell voltage-clamp. I(Ca) was increased in afternoon vs. morning in GnRH neurons from OVXE mice but this increase was abolished in cells from OVXEP mice. I(Ca) in cells from OVXD mice was increased regardless of time of day; there was no additional effect in OVXED mice. P reduced N-type and DHT potentiated N- and R-type VGCCs; P blocked the DHT potentiation of N-type-mediated current. These data suggest P and DHT have opposing actions on VGCCs in GnRH neurons, but in the presence of both steroids, P dominates. VGCCs are targets of ovarian steroid feedback modulation of GnRH neuron activity and, more specifically, a potential mechanism whereby androgens could activate GnRH neuronal function.

Inhibition of aldehyde dehydrogenase-2 suppresses cocaine seeking by generating THP, a cocaine use–dependent inhibitor of dopamine synthesis

There is no effective treatment for cocaine addiction despite extensive knowledge of the neurobiology of drug addiction. Here we show that a selective aldehyde dehydrogenase-2 (ALDH-2) inhibitor, ALDH2i, suppresses cocaine self-administration in rats and prevents cocaine- or cue-induced reinstatement in a rat model of cocaine relapse-like behavior. We also identify a molecular mechanism by which ALDH-2 inhibition reduces cocaine-seeking behavior: increases in tetrahydropapaveroline (THP) formation due to inhibition of ALDH-2 decrease cocaine-stimulated dopamine production and release in vitro and in vivo. Cocaine increases extracellular dopamine concentration, which activates dopamine D2 autoreceptors to stimulate cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) in primary ventral tegmental area (VTA) neurons. PKA and PKC phosphorylate and activate tyrosine hydroxylase, further increasing dopamine synthesis in a positive-feedback loop. Monoamine oxidase converts dopamine to 3,4-dihydroxyphenylacetaldehyde (DOPAL), a substrate for ALDH-2. Inhibition of ALDH-2 enables DOPAL to condense with dopamine to form THP in VTA neurons. THP selectively inhibits phosphorylated (activated) tyrosine hydroxylase to reduce dopamine production via negative-feedback signaling. Reducing cocaine- and craving-associated increases in dopamine release seems to account for the effectiveness of ALDH2i in suppressing cocaine-seeking behavior. Selective inhibition of ALDH-2 may have therapeutic potential for treating human cocaine addiction and preventing relapse.

Tumor necrosis factor-α: a reno-protective cytokine? Focus on “Tumor necrosis factor-α suppresses angiotensinogen expression through formation of a p50/p50 homodimer in human renal proximal tubular cells”

<i>Tumor necrosis factor-α: a reno-protective cytokine?</i> Focus on “Tumor necrosis factor-α suppresses angiotensinogen expression through formation of a p50/p50 homodimer in human renal proximal tubular cells”

Regulatory mechanism of hypothalamo-pituitary–adrenal (HPA) axis and neuronal changes after adrenalectomy in type 2 diabetes

Diabetes, especially type 2, is closely associated with hypothalamo-pituitary–adrenal (HPA) axis regulation. Short-term effects of adrenalectomy (ADX) in type 2 diabetes are well characterized; however, there have been few reports on the long-term effects of ADX in genetically engineered type 2 diabetes and the neuroendocrine system. We performed bilateral ADX in Zucker Lean Control rats (ZLC; ADX-ZLC), Zucker Diabetic Fatty rats (ZDF; ADX-ZDF), and sham control rats to evaluate how the HPA axis would be regulated in long-term corticosterone deficient type 2 diabetic animals. We evaluated arginine vasopressin (AVP), glucocorticoid receptor (GR), and corticotropin-releasing hormone (CRH) expression with immunohistochemistry (IHC), immunofluorescence, real-time PCR, and Western blot analysis in each treatment group 7 weeks post ADX to assess HPA axis regulatory patterns in connection with type 2 diabetes. Additionally, mRNA expression of AVP and CRH receptors (V1aR, V1bR, CRHR1, and CRHR2) was also measured and adrenocorticotropin hormone (ACTH) immunoreactivity was surveyed by IHC to add to data regarding the regulatory mechanism. AVP and CRH protein expression levels increased after ADX in the hypothalamus of diabetic rats based on IHC results; however, we found that the subtypes of each receptor may be regulated differently in ADX groups compared to sham groups. Immunoreactivity of ACTH in the pituitary gland was enhanced in ADX groups and GR expression levels in the hypothalamic paraventricular nuclei (PVN) remained high, as determined by IHC as well as Western blot analysis. Without the negative feedback system of corticosterone, CRH is highly enhanced and may primarily combine with CRHR1 to stimulate negative feedback through ACTH in the pituitary gland in type 2 diabetic rats with long-term ADX. Although the negative feedback signal was not transmitted appropriately following long-term ADX with type 2 diabetes, a high GR protein level was maintained as in type 2 diabetes. The long-termed lack of corticosterone in the blood stream is a very important factor for normal regulation of the HPA axis even in diabetic animals. From the data, we can conclude that the stimulated HPA axis regulation in the developing type 2 diabetic animals following long-term adrenalectomy has remained elevated rather than diminished. Therefore, the current study may provide useful information to better understand patients suffering from both type 2 diabetes and Addison's disease.

Insulin like growth factor binding protein-7 reduces growth of human breast cancer cells and xenografted tumors

Previously, we have shown that insulin-like growth factor binding protein-7 (IGFBP-7) expression is inversely correlated with disease progression in breast cancer and is associated with poor outcome. To further investigate the role of IGFBP-7 in the growth and metastatic behavior of breast cancer, primary breast tumors and metastatic tumors derived from the same patients were analyzed for IGFBP-7 expression. Immunohistochemical analysis revealed that IGFBP-7 is downregulated in half of the human metastatic breast tumors tested. IGFBP-7 has been linked to suppression of oncogenic pathways and can directly restore cellular senescence in melanomas, leading to their regression. It is possible that breast tumors with metastatic potential have escaped from IGFBP-7-induced suppression by its down-regulation. Twenty-two human primary breast tumor specimens were transplanted into human-bone NOD/SCID mice. One of the two triple negative primary breast tumors was serially xenotransplanted more than five times. Each serial transplant resulted in increased tumor take and rate of growth. Expression of IGFBP-7 was downregulated upon each serial implantation. To investigate the role of IGFBP-7 in breast tumor suppression, IGFBP-7 was overexpressed in the triple negative MDA-MB-468 human breast cancer line by stable transfection of a pSec-tag2-IGFBP-7 vector. The parental MDA-MB-468 breast cancer cells expressed extremely low levels of endogenous IGFBP-7. The production of IGFBP-7 protein by the MDA-MB-468 cells stably transfected with IGFBP-7 was confirmed by immunoblotting with anti-IGFBP-7 antibody. Ectopic overexpression of IGFBP-7 significantly reduced the growth of the IGFBP-7 transfected MDA-MB-468 cells compared to the parental MDA-MB-468 cells. We also assessed the role of IGFBP-7 on cell migration, a key determinant of malignant progression and metastasis. When parental MDA-MB-468 cells were treated with various amounts of conditioned medium derived from the IGFBP-7 overexpressing cell line, a significant difference in cell migration rate was observed between untreated and treated cells. IGFBP-7 strongly suppressed the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK-1/2, suggesting that IGFBP-7 mediates its anti-proliferative effects through negative feedback signaling. Levels of phospho-ERK-1/2 were higher in the parental MDA-MB-468 than in IGFBP-7-expressing cells derived from it. When injected subcutaneously into NOD/SCID mice, the increased expression of IGFBP-7 in the MDA-MB-468 transfected cells reduced the rate of tumor growth in comparison to the parental MDA-MB-468 controls. These results suggest that the growth of breast cancer could be prevented by the forced expression of IGFBP-7 protein.

The ADPKD genes pkd1a/b and pkd2 regulate extracellular matrix formation

Mutations in polycystin1 (PKD1) account for the majority of autosomal dominant polycystic kidney disease (ADPKD). PKD1 mutations are also associated with vascular aneurysm and abdominal wall hernia, suggesting a role for polycystin1 in extracellular matrix (ECM) integrity. In zebrafish, combined knockdown of the PKD1 paralogs pkd1a and pkd1b resulted in dorsal axis curvature, hydrocephalus, cartilage and craniofacial defects, and pronephric cyst formation at low frequency (10-15%). Dorsal axis curvature was identical to the axis defects observed in pkd2 knockdown embryos. Combined pkd1a/b, pkd2 knockdown demonstrated that these genes interact in axial morphogenesis. Dorsal axis curvature was linked to notochord collagen overexpression and could be reversed by knockdown of col2a1 mRNA or chemical inhibition of collagen crosslinking. pkd1a/b- and pkd2-deficient embryos exhibited ectopic, persistent expression of multiple collagen mRNAs, suggesting a loss of negative feedback signaling that normally limits collagen gene expression. Knockdown of pkd1a/b also dramatically sensitized embryos to low doses of collagen-crosslinking inhibitors, implicating polycystins directly in the modulation of collagen expression or assembly. Embryos treated with wortmannin or LY-29400 also exhibited dysregulation of col2a1 expression, implicating phosphoinositide 3-kinase (PI3K) in the negative feedback signaling pathway controlling matrix gene expression. Our results suggest that pkd1a/b and pkd2 interact to regulate ECM secretion or assembly, and that altered matrix integrity may be a primary defect underlying ADPKD tissue pathologies.

The ADPKD genes pkd1a/b and pkd2 regulate extracellular matrix formation

Mutations in polycystin1 (PKD1) account for the majority of autosomal dominant polycystic kidney disease (ADPKD). PKD1 mutations are also associated with vascular aneurysm and abdominal wall hernia, suggesting a role for polycystin1 in extracellular matrix (ECM) integrity. In zebrafish, combined knockdown of the PKD1 paralogs pkd1a and pkd1b resulted in dorsal axis curvature, hydrocephalus, cartilage and craniofacial defects, and pronephric cyst formation at low frequency (10-15%). Dorsal axis curvature was identical to the axis defects observed in pkd2 knockdown embryos. Combined pkd1a/b, pkd2 knockdown demonstrated that these genes interact in axial morphogenesis. Dorsal axis curvature was linked to notochord collagen overexpression and could be reversed by knockdown of col2a1 mRNA or chemical inhibition of collagen crosslinking. pkd1a/b- and pkd2-deficient embryos exhibited ectopic, persistent expression of multiple collagen mRNAs, suggesting a loss of negative feedback signaling that normally limits collagen gene expression. Knockdown of pkd1a/b also dramatically sensitized embryos to low doses of collagen-crosslinking inhibitors, implicating polycystins directly in the modulation of collagen expression or assembly. Embryos treated with wortmannin or LY-29400 also exhibited dysregulation of col2a1 expression, implicating phosphoinositide 3-kinase (PI3K) in the negative feedback signaling pathway controlling matrix gene expression. Our results suggest that pkd1a/b and pkd2 interact to regulate ECM secretion or assembly, and that altered matrix integrity may be a primary defect underlying ADPKD tissue pathologies.

Abstract 5075: PI3K activation restores KrasG12D oncogenic ability in a mouse model of follicular thyroid cancer by directly opposing multiple Kras-initiated negative feedback signals

Abstract Activating mutations of Ras family members and aberrant activation of the PI3K/AKT pathway are quite common in thyroid cancer originating from the follicular epithelium. We have recently shown in a relevant in vivo model that, contrary to many other cell types, Kras activation is unable to induce oncogenic transformation of the thyroid follicular cells. However, PI3K activation is able to potently cooperate with Kras oncogenic mutation, thus inducing aggressive and metastatic thyroid follicular carcinomas. We now show that one key event mediated by PI3K in this cross-talk is the inhibition of multiple Kras-initiated negative feedback signals shutting down MAPK signaling and the induction of proliferation. Through these mechanisms, PI3K signaling restores Kras-initiated MAPK activation, induction of proliferation, and oncogenic transformation. In particular, our data reveal that activation of p38α MAPK, as well as up-regulation of MKP2 and MKP3 phosphatases, play a pivotal role in mediating the growth-suppressive negative feedback initiated by Kras oncogenic activation. Activation of p38α MAPK acts by inducing cell cycle arrest and the establishment of cellular senescence, while MKP2 and MKP3 stunt ERK1/2 activation through direct dephosphorylation of these kinases. Strikingly, we have found that PI3K acts through Akt1 (but not Akt2) to potently suppress p38 phosphorylation and activation. Furthermore, PI3K activation results in the transcriptional repression of MKP2 and MKP3 promoters. Through these two molecular mechanisms, PI3K activation contributes to unleashing to its full extent Kras oncogenic ability. These data not only uncover novel oncogenic pathways controlled by PI3K signaling, but also further strengthen the rationale for the dual targeting of PI3K and MAPK pathways in thyroid cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5075.

Abstract 1653: Highly effective treatment of HER2-amplified breast cancers through the complete inactivation of the HER2-HER3 tumorigenic driver

Abstract Approximately 25% of breast cancers are driven by amplification of the HER2 oncogene and over-activity of the HER2-HER3 signaling complex. It is a promising hypothesis that the pharmacologic inactivation of HER2-HER3 signaling with tyrosine kinase inhibitors (TKIs) in patients will be a highly effective treatment for this disease. Treatment of HER2 amplified breast cancers with HER2 targeting agents shows that the HER2-HER3 complex is endowed with a two-log signal buffering capacity, making drug potency a critical and potentially limiting variable. The buffering capacity is mediated through Akt driven negative feedback signaling, which has the ability to amplify HER2-HER3 signaling and protect it against incomplete inhibition of HER2 kinase. The concomitant use of downstream inhibitors induces compound sensitizing and desensitizing mechanisms and incremental anti-tumor effects. The HER2-HER3 signal buffering mechanisms cannot ultimately compensate for the total loss of HER2 catalytic activity, and at fully inactivating doses of TKI, the failure to phosphorylate HER3 and the dual loss of MAPK and Akt pathway signaling leads to apoptotic tumor cell death. Such HER2-inactivating dosing is not tolerable in vivo using continuous schedules but is feasible and highly effective using intermittent dosing. We show that continuous dosing of the HER2 TKI, lapatinib, at its maximum tolerated dose in mice bearing HER2-amplified tumors produces modest growth inhibitory effects. However, a five day repeated intermittent schedule allows eight fold higher dosing and produces much more profound tumor regression and durable anti-tumor effects. Therefore high dose intermittent TKI therapy is an imminently testable clinical treatment hypothesis that can fully inactivate HER2-HER3 signaling by overpowering its signal buffering capacity, revealing a monotherapy potential in HER2-targeting TKIs that may surpass many of the combination therapy approaches currently being explored. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1653.

Abstract 5588: T cell activation in relation to clinical responsiveness in patients with metastatic hormone-refractory prostate cancer receiving combined Prostate GVAX and anti-CTLA4 immunotherapy

Abstract The effects of a GM-CSF-secreting allogeneic prostate cancer vaccine (Prostate GVAX) and the anti-CTLA4 antibody Ipilimumab were investigated in a Phase I dose escalation/expansion trial of patients with metastatic, hormone-refractory prostate cancer (mHRPC). Patients received a 500 million cell GVAX priming dose on day 1 followed by 12 bi-weekly intradermal administrations of 300 million cells, while Ipilimumab was administered every 4 wks from day 1 for a total of 6 times. Initially, 12 patients were enrolled in cohorts of 3 and each cohort was assigned an escalating dose of Ipilimumab at 0.3, 1, 3 or 5 mg/kg. 16 additional patients were enrolled in the expansion cohort of 3 mg/kg Ipilimumab. Results showed that the GVAX and Ipilimumab combination was clinically active in mHRPC patients. PSA declines of more than 50% (Partial Response, PR) were observed in 5 of 22 patients in the 3-5 mg/kg Ipilimumab doses and PSA stabilizations (Stable Disease, SD) were observed in 1/3 pts in lower (0.3 and 1 mg/kg), and in 7/22 in the higher (3-5 mg/kg) dose levels. Phenotypic and functional (anti-tumor) effector T cell activation was monitored to identify changes that might predict or correlate with clinical efficacy. Most notably, pronounced and significant increases in frequencies of activated and effector CD4+ and CD8+ T cells were observed by HLA-DR and ICOS expression upon administration of high (3 and 5 mg/kg) but not of low (0.3 and 1 mg/kg) Ipilimumab doses. We also observed a significant increase of CTLA4 and PD-1 expression on CD4+ T cells compared to pre-treatment values in the 3 mg/kg Ipilimumab-administered patients. The latter might be indicative of the induction of concomitant negative feedback signaling. As an indiciation of Tumor-Associated Antigen (TAA) specific responsiveness we tested HLA-Tetramer (Tm) reactivity to NY-ESO and PSMA in HLA-A2 or -A3 positive patients. For NY-ESO, GVAX/Ipi-induced increased seroreactivity (by Western Blot or ELISA) was observed in 6/28 patients. Of these 6 patients, three could be tested for Tm reactivity and in two (1SD, 1 Progressive Disease, PD) increased NY-ESO157 rates were found concomitant with increasing serum Ab levels, whereas no Tm reactivity was detected in 8 patients without seroconversions. Whereas PSMA seroconversions were observed in a total of 12/28 patients (and of note, in 4/5 PR), no Tm positivity at any time was found in a total of 15 patients tested. Further functional Treg and CTL analyses are ongoing. So far, only early HLA-DR upregulation on CD4+ or CD8+ T cells (visit 1 versus visit 3) showed any value for response prediction, since it was observed to significant levels in patients with PR or SD, but not in patients with PD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5588.

Extracellular adenosine triggers lymphocyte entry into the central nervous system during experimental autoimmune encephalomyelitis by regulating chemokine and adhesion molecule expression in the brain (44.12)

Abstract Lymphocyte entry into the central nervous system (CNS) is a highly regulated process which serves to promote tissue repair while protecting against potentially fatal inflammation. As the formation of extracellular adenosine (catalyzed from ATP by CD39 and CD73) during inflammation serves as a negative-feedback signal to prevent excessive cell damage, our results suggest that adenosine receptor (AR) signaling also regulates CNS lymphocyte infiltration. We have shown that extracellular adenosine is required for the progression experimental autoimmune encephalomyelitis (EAE), the animal model of autoimmune inflammatory disease Multiple Sclerosis. We now show that AR signaling induces the CNS expression of the chemokine CX3CL1 (fractalkine), which acts as a chemoattractant for monocytes, lymphocytes, and NK cells. In addition, CX3CL1 expression is up-regulated in the CNS immediately prior to EAE onset only in wild type, but not CD73-/- mice, which cannot produce extracellular adenosine, are protected from EAE, and have minimal CNS lymphocyte infiltration following EAE induction. Moreover, CD39, CD73, and ARs are found clustered in the brain only at the choroid plexus, a structure which is known to regulate lymphocyte migration into the CNS. As AR signaling can induce CX3CL1 and ICAM-1 expression and lymphocyte transmigration across an in vitro choroid plexus transwell barrier, we conclude that AR signaling regulates lymphocyte entry into the CNS.

The ADPKD genespkd1a/bandpkd2regulate extracellular matrix formation

Mutations in polycystin1 (PKD1) account for the majority of autosomal dominant polycystic kidney disease (ADPKD). PKD1 mutations are also associated with vascular aneurysm and abdominal wall hernia, suggesting a role for polycystin1 in extracellular matrix (ECM) integrity. In zebrafish, combined knockdown of the PKD1 paralogs pkd1a and pkd1b resulted in dorsal axis curvature, hydrocephalus, cartilage and craniofacial defects, and pronephric cyst formation at low frequency (10-15%). Dorsal axis curvature was identical to the axis defects observed in pkd2 knockdown embryos. Combined pkd1a/b, pkd2 knockdown demonstrated that these genes interact in axial morphogenesis. Dorsal axis curvature was linked to notochord collagen overexpression and could be reversed by knockdown of col2a1 mRNA or chemical inhibition of collagen crosslinking. pkd1a/b- and pkd2-deficient embryos exhibited ectopic, persistent expression of multiple collagen mRNAs, suggesting a loss of negative feedback signaling that normally limits collagen gene expression. Knockdown of pkd1a/b also dramatically sensitized embryos to low doses of collagen-crosslinking inhibitors, implicating polycystins directly in the modulation of collagen expression or assembly. Embryos treated with wortmannin or LY-29400 also exhibited dysregulation of col2a1 expression, implicating phosphoinositide 3-kinase (PI3K) in the negative feedback signaling pathway controlling matrix gene expression. Our results suggest that pkd1a/b and pkd2 interact to regulate ECM secretion or assembly, and that altered matrix integrity may be a primary defect underlying ADPKD tissue pathologies.

A Negative Feedback Signal That Is Triggered by Peril Curbs Honey Bee Recruitment

Decision making in superorganisms such as honey bee colonies often uses self-organizing behaviors, feedback loops that allow the colony to gather information from multiple individuals and achieve reliable and agile solutions. Honey bees use positive feedback from the waggle dance to allocate colony foraging effort. However, the use of negative feedback signals by superorganisms is poorly understood. I show that conspecific attacks at a food source lead to the production of stop signals, communication that was known to reduce waggle dancing and recruitment but lacked a clear natural trigger. Signalers preferentially targeted nestmates visiting the same food source, on the basis of its odor. During aggressive food competition, attack victims increased signal production by 43 fold. Foragers that attacked competitors or experienced no aggression did not alter signal production. Biting ambush predators also attack foragers at flowers. Simulated biting of foragers or exposure to bee alarm pheromone also elicited signaling (88-fold and 14-fold increases, respectively). This provides the first clear evidence of a negative feedback signal elicited by foraging peril to counteract the positive feedback of the waggle dance. As in intra- and intercellular communication, negative feedback may play an important, though currently underappreciated, role in self-organizing behaviors within superorganisms.

Synaptic localization of the enzymatic machinery responsible for 2-arachidonoylglycerol synthesis and degradation in the human hippocampus

Event Abstract Back to Event Synaptic localization of the enzymatic machinery responsible for 2-arachidonoylglycerol synthesis and degradation in the human hippocampus Anikó Ludányi1*, Zsófia Maglóczky1, Ken Mackie2, Tamás Freund1 and István Katona1 1 Hungarian Academy of Sciences, Institute of Experimental Medicine, Hungary 2 Indiana University, Department of Psychological and Brain Sciences, United States Endocannabinoid signaling is involved in several forms of synaptic plasticity in the brain. Although molecular architecture of the endocannabinoid system has already been revealed in rodents, whether it is an evolutionarily conserved feature of hippocampal excitatory synapses has remained elusive. To address this issue, we studied the subcellular localization of enzymes synthesizing and degrading endocannabinoid 2-arachidonoylglycerol (2-AG) in the human hippocampus (n = 6). At the light microscopic level, immunostaining for diacylglycerol lipase-alpha (DGL-alpha), the main biosynthetic enzyme of 2-AG, visualized a similar staining pattern in post mortem human hippocampus as found earlier in the rodent. At higher magnification, DGL-alpha-immunopositive puncta throughout the neuropil outlined immunonegative main apical dendrites of pyramidal cells. Further electron microscopic investigations revealed that labeling corresponds to the accumulation of DGL-alpha in dendritic spine heads. Notably, immunostaining for 2-AG’s predominant degrading enzyme, monoacylglycerol lipase (MGL) resulted in very similar staining pattern at the light microscopic level. However, further electron microscopic analysis revealed that MGL was enriched in axon terminals, but not in spine heads. Our results suggest that formation of 2-AG by DGL-alpha occurs in postsynaptic dendritic spines, and after acting on presynaptic CB1 cannabinoid receptors, it is degraded in axon terminals by MGL. Taken together, these findings show that the localization of metabolic enzymes involved in synaptic 2-AG signaling is similar in rodent and human suggesting an evolutionarily conserved feature of excitatory synapses. Moreover, the molecular architecture of endocannabinoid signaling seems to be precisely designed to subserve retrograde regulation of synaptic neurotransmission and likely has a fundamental physiological role as a negative feed-back signal protecting synapses from excessive presynaptic activity. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Cellular neuroscience Citation: Ludányi A, Maglóczky Z, Mackie K, Freund T and Katona I (2010). Synaptic localization of the enzymatic machinery responsible for 2-arachidonoylglycerol synthesis and degradation in the human hippocampus. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00091 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 22 Apr 2010; Published Online: 22 Apr 2010. * Correspondence: Anikó Ludányi, Hungarian Academy of Sciences, Institute of Experimental Medicine, Budapest, Hungary, ludanyi@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Anikó Ludányi Zsófia Maglóczky Ken Mackie Tamás Freund István Katona Google Anikó Ludányi Zsófia Maglóczky Ken Mackie Tamás Freund István Katona Google Scholar Anikó Ludányi Zsófia Maglóczky Ken Mackie Tamás Freund István Katona PubMed Anikó Ludányi Zsófia Maglóczky Ken Mackie Tamás Freund István Katona Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Single-cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response.

Understanding how the immune system decides between tolerance and activation by antigens requires addressing cytokine regulation as a highly dynamic process. We quantified the dynamics of interleukin-2 (IL-2) signaling in a population of T cells during an immune response by combining in silico modeling and single-cell measurements in vitro. We demonstrate that IL-2 receptor expression levels vary widely among T cells creating a large variability in the ability of the individual cells to consume, produce and participate in IL-2 signaling within the population. Our model reveals that at the population level, these heterogeneous cells are engaged in a tug-of-war for IL-2 between regulatory (T(reg)) and effector (T(eff)) T cells, whereby access to IL-2 can either increase the survival of T(eff) cells or the suppressive capacity of T(reg) cells. This tug-of-war is the mechanism enforcing, at the systems level, a core function of T(reg) cells, namely the specific suppression of survival signals for weakly activated T(eff) cells but not for strongly activated cells. Our integrated model yields quantitative, experimentally validated predictions for the manipulation of T(reg) suppression.

Testosterone: More Is Not Always Better

Having conducted population-based studies on the relationship of sex steroid levels to bone mass in Rochester, Minnesota for many years, I have been struck by just how often male members of the medical staff at our institution who participate in these studies inquire about their testosterone level. Discovering that they happen to be in the top 10th percentile is often a cause for celebration; conversely, being told that they happen to be “average” or, most distressingly, “below average,” generally leads to at least temporary anguish. Not surprisingly, despite the numerous publications from our group and others regarding the key role for estrogen in bone metabolism in men (summarized in Ref. 1), none of my colleagues inquire or seem to care one way or another about their estradiol level. At least based on this anecdotal experience, it seems that even in the minds of well-informed male medical professionals, more testosterone is clearly “better,” whereas estrogen is largely irrelevant. It is in this context that men with modest testosterone levels should perhaps have their chance to celebrate based on the results of the study by Burnett-Bowie et al. (2) published in this issue of JCEM. This carefully conducted clinical trial clearly demonstrates that at least for bone, more testosterone is not necessarily better, especially if it comes at the price of lower estrogen production. The trial is based on the premise that because male aging is associated with declining testosterone levels and with increased body fat, decreased muscle mass, strength, and bone mineral density (BMD) (1), replacing testosterone in elderly men should be beneficial. However, rather than administering exogenous testosterone, which requires topical or parenteral dosing and significantly increases estrogen levels (via peripheral aromatization) and which may, in turn, result in side effects such as gynecomastia, the authors chose to test the effects of increasing endogenous testosterone production by blocking aromatization of testosterone to estradiol using a potent orally administered aromatase inhibitor, anastrozole (2). By removing negative feedback signals, the anastrozole-induced decrease in estradiol increases GnRH and gonadotropin production at the hypothalamus and pituitary, respectively, thereby increasing testosterone production (3). The Burnett-Bowie et al. (2) paper reports on 69 men, aged 60 and older, who completed the 1-yr, double-blind, randomized, placebo-controlled trial. Anastrozole had the desired effects on serum testosterone levels, with mean values increasing from 319 ng/dl (the lower end of the normal range) at baseline to 524 ng/dl at 3 months and a slight decline to 474 ng/dl at 1 yr, an overall increase of approximately 50%. As expected, estradiol levels decreased (by 20%), from 15 to 12 pg/ml at 3 months, and remained stable at this level thereafter. However, despite the substantial increase in serum testosterone levels, posterior-anterior (PA) spine BMD decreased by 1.7% in the anastrozole group while increasing by 0.8% in the placebo group (P 0.0014 for the difference between groups), with similar trends for differences between the groups for femoral neck, total hip, and total body BMD. The inescapable conclusion from these data is that the 50% increase in testosterone levels was trumped by the much more modest 20% reduction in estradiol levels, leading to negative skeletal effects of anastrozole therapy in aging men. To some extent, these findings are not surprising and are perhaps predictable, based on the now extensive body of evidence for a critical role for estrogen in regulating the male skeleton (1). Although previous studies in men had shown that serum estradiol levels were related to bone density (4) and that estrogen regulated bone turnover (5,

Severe Early-Onset Inflammatory Bowel Disease Caused by IL10 Receptor Deficiency Can Be Cured by Allogeneic Hematopoietic Stem Cell Transplantation.

Abstract 713In spite of recent genome-wide association studies, the molecular pathophysiology of many human auto-inflammatory diseases such as enterocolitis remains largely unknown.Here, we discover the first fully penetrant monogenetic defect causing inflammatory bowel disease (IBD) in humans. Using homozygosity mapping and candidate gene sequencing, we identified three distinct, homozygous mutations in IL10RA, encoding the IL10R1 protein, and IL10RB, encoding the IL10R2 protein, in patients with severe and refractory enterocolitis. IL10R1 is a specific receptor for IL10, whereas IL10R2 is a shared cytokine receptor unit for IL10, IL22, IL26, and IFNλ. The striking similarity of the clinical phenotype between patients with IL10RA and IL10RB deficiency, respectively, suggests that defective IL10-mediated signaling, and not IL22, IL26, or IFNλ dependent effects, is the critical reason for disease.Deleterious missense mutations in IL10RA abrogate IL10-induced signaling, as shown by deficient phosphorylation of STAT3 at the residue tyrosine 705 in primary patient cells and in HeLa cells engineered to express mutant IL10R1. Mutations in IL10RB introduced a premature stop codon. Defective expression of IL10R2 on the cell surface and deficient STAT3 signaling could be reconstituted by lentiviral gene transfer. As a consequence of defective STAT3 signal transduction in response to IL10 stimulation, IL10R-deficient primary cells showed increased secretion of TNFαa and various other proinflammatory cytokines unresponsive to IL10-dependent negative feedback regulation (TGFβ1, IL1αa, IL1β, IL2, IL6, IL6sR, RANTES, MCP1, MIP1αa, and MIP1β). We are currently assessing whether other IBD patients with early-onset IBD show defects in IL10-mediated signal transduction.In view of the therapy refractory course of disease and the critical role of IL10 signaling on cells of the hematopoietic system, we have successfully treated two IBD patients with IL10 receptor deficiency by hematopoietic stem cell transplantation (HSCT) without overt side effects. This proof-of-principle suggests that allogeneic HSCT may represent a novel therapeutic approach to treat defined subgroups of IBD patients.In summary, our results suggest that IL10 receptor defects constitute monogenetic causes for severe, early-onset IBD patients, proving that a lack of IL10-mediated negative feedback signaling perturbs homeostasis of the intestinal immune system. Disclosures:No relevant conflicts of interest to declare.