Presynaptic NMDA receptors mediate IPSC potentiation at GABAergic synapses in developing rat neocortex

Background

NMDA receptors are traditionally viewed as being located postsynaptically, at both synaptic and extrasynaptic locations. However, both anatomical and physiological studies have indicated the presence of NMDA receptors located presynaptically. Physiological studies of presynaptic NMDA receptors on neocortical GABAergic terminals and their possible role in synaptic plasticity are lacking.

Methodology/Principal Findings

We report here that presynaptic NMDA receptors are present on GABAergic terminals in developing (postnatal day (PND) 12-15) but not older (PND21-25) rat frontal cortex. Using MK-801 in the recording pipette to block postsynaptic NMDA receptors, evoked and miniature IPSCs were recorded in layer II/III pyramidal cells in the presence of AMPA/KA receptor antagonists. Bath application of NMDA or NMDA receptor antagonists produced increases and decreases in mIPSC frequency, respectively. Physiologically patterned stimulation (10 bursts of 10 stimuli at 25 Hz delivered at 1.25 Hz) induced potentiation at inhibitory synapses in PND12-15 animals. This consisted of an initial rapid, large increase in IPSC amplitude followed by a significant but smaller persistent increase. Similar changes were not observed in PND21-25 animals. When 20 mM BAPTA was included in the recording pipette, potentiation was still observed in the PND12-15 group indicating that postsynaptic increases in calcium were not required. Potentiation was not observed when patterned stimulation was given in the presence of D-APV or the NR2B subunit antagonist Ro25-6981.

Conclusions/Significance

The present results indicate that presynaptic NMDA receptors modulate GABA release onto neocortical pyramidal cells. Presynaptic NR2B subunit containing NMDA receptors are also involved in potentiation at developing GABAergic synapses in rat frontal cortex. Modulation of inhibitory GABAergic synapses by presynaptic NMDA receptors may be important for proper functioning of local cortical networks during development.     

Phylogenetic relationships of Hepatozoon (Apicomplexa: Adeleorina) based on molecular, morphologic, and life-cycle characters

To evaluate higher-level affinities of Hepatozoon species within Apicomplexa, we sequenced the 18S rRNA gene from 2 parasites (Hepatozoon americanum and Hepatozoon canis) of dogs and 1 (Hepatozoon catesbianae) of bullfrogs. Sequences from other apicomplexans among the Sarcocystiidae, Eimeriidae, Theileriidae, Plasmodiidae, Cryptosporiidae, and Babesiidae, a Perkinsus species and 2 dinoflagellates were obtained from GenBank. Phylogenetic analysis indicated that Plasmodium, Cryptosporidium, and Hepatozoon form a monophyletic group distinct from representatives of other apicomplexan families. Although equivocal, our analysis indicated that Plasmodium and Cryptosporidium are sister taxa and that Hepatozoon is basal to them. To evaluate phylogenetic affinities among H. americanum, H. canis, and other species of Hepatozoon, we examined 18 morphologic and life-cycle features of 13 species currently assigned to Hepatozoon. This analysis indicates paraphyly of Hepatozoon (as currently arranged) because Hepatozoon lygosomarum was found most closely related to Hemolivia mauritanicum. These results, combined with results of previous studies, support elevating Hepatozoon to familial level (Hepatozoidae) as originally suggested by Wenyon in 1926. Both DNA sequence data and morphologic and life-cycle characters support a sister-group relationship between H. americanum and H. canis.     

Understanding and modeling alternating tangential flow filtration for perfusion cell culture

Alternating tangential flow (ATF) filtration has been used with success in the Biopharmaceutical industry as a lower shear technology for cell retention with perfusion cultures. The ATF system is different than tangential flow filtration; however, in that reverse flow is used once per cycle as a means to minimize fouling. Few studies have been reported in the literature that evaluates ATF and how key system variables affect the rate at which ATF filters foul. In this study, an experimental setup was devised that allowed for determination of the time it took for fouling to occur for given mammalian (PER.C6) cell culture cell densities and viabilities as permeate flow rate and antifoam concentration was varied. The experimental results indicate, in accordance with D'Arcy's law, that the average resistance to permeate flow (across a cycle of operation) increases as biological material deposits on the membrane. Scanning electron microscope images of the post‐run filtration surface indicated that both cells and antifoam micelles deposit on the membrane. A unique mathematical model, based on the assumption that fouling was due to pore blockage from the cells and micelles in combination, was devised that allowed for estimation of sticking factors for the cells and the micelles on the membrane. This model was then used to accurately predict the increase in transmembane pressure during constant flux operation for an ATF cartridge used for perfusion cell culture. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1291–1300, 2014     

HIF1-alpha functions as a tumor promoter in cancer associated fibroblasts,and as a tumor suppressor in breast cancer cells: Autophagy drives compartment-specific oncogenesis

Our recent studies have mechanistically implicated a loss of stromal Cav-1 expression and HIF1α-activation in driving the cancer-associated fibroblast phenotype, through the paracrine production of nutrients via autophagy and aerobic glycolysis. However, it remains unknown if HIF1α-activation is sufficient to confer the cancer-associated fibroblast phenotype. To test this hypothesis directly, we stably-expressed activated HIF1α in fibroblasts and then examined their ability to promote tumor growth using a xenograft model employing human breast cancer cells (MDA-MB-231). Fibroblasts harboring activated HIF1α showed a dramatic reduction in Cav-1 levels and a shift towards aerobic glycolysis, as evidenced by a loss of mitochondrial activity, and an increase in lactate production. Activated HIF1α also induced BNIP3 and BNIP3L expression, markers for the autophagic destruction of mitochondria. Most importantly, fibroblasts expressing activated HIF1α increased tumor mass by ∼2-fold and tumor volume by ∼3-fold, without a significant increase in tumor angiogenesis. In this context, HIF1α also induced an increase in the lymph node metastasis of cancer cells. Similar results were obtained by driving NFκB activation in fibroblasts, another inducer of autophagy. Thus, activated HIF1α is sufficient to functionally confer the cancer-associated fibroblast phenotype. It is also known that HIF1α expression is required for the induction of autophagy in cancer cells. As such, we next directly expressed activated HIF1α in MDA-MB-231 cells and assessed its effect on tumor growth via xenograft analysis. Surprisingly, activated HIF1α in cancer cells dramatically suppressed tumor growth, resulting in a 2-fold reduction in tumor mass and a three-fold reduction in tumor volume. We conclude that HIF1α activation in different cell types can either promote or repress tumorigenesis. Based on these studies, we suggest that autophagy in cancer-associated fibroblasts promotes tumor growth via the paracrine production of recycled nutrients, which can directly “feed” cancer cells. Conversely, autophagy in cancer cells represses tumor growth via their “self-digestion.” Thus, we should consider that the activities of various known oncogenes and tumor-suppressors may be compartment and cell-type specific, and are not necessarily an intrinsic property of the molecule itself. As such, other “classic” oncogenes and tumor suppressors will have to be re-evaluated to determine their compartment specific effects on tumor growth and metastasis. Lastly, our results provide direct experimental support for the recently proposed “autophagic tumor stroma model of cancer.”Key words: caveolin-1, autophagy, mitophagy, the Warburg effect, tumor stroma, hypoxia, HIF1A, NFκB, compartment-specific oncogenesis, cancer-associated fibroblasts     

Localization of ERK/MAP kinase is regulated by the alphaherpesvirus tegument protein Us2

Many different viruses activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase signaling pathway during infection and require ERK activation for the efficient execution of their replication programs. Despite these findings, no virus-encoded proteins have been identified that directly modulate ERK activities. In an effort to determine the function of a conserved alphaherpesvirus structural protein called Us2, we screened a yeast two-hybrid library derived from NIH 3T3 cells and identified ERK as a Us2-interacting protein. Our studies indicate that Us2 binds to ERK in virus-infected cells, mediates the incorporation of ERK into the virion, and inhibits the activation of ERK nuclear substrates. The association of Us2 with ERK leads to the sequestration of ERK at the plasma membrane and to a perinuclear vesicular compartment, thereby keeping ERK out of the nucleus. Us2 can bind to activated ERK, and the data suggest that Us2 does not inhibit ERK enzymatic activity. The treatment of cells with U0126, a specific inhibitor of ERK activation, resulted in a substantial delay in the release of virus from infected cells that was more pronounced with a virus deleted for Us2 than with parental and repaired strains, suggesting that both ERK and Us2 activities are required for efficient virus replication. This study highlights an additional complexity to the activation of ERK by viruses, namely, that localization of active ERK can be altered by virus-encoded proteins.     

Inhibition of integrin-mediated crosstalk with epidermal growth factor receptor/Erk or Src signaling pathways in autophagic prostate epithelial cells induces caspase-independent death

In vivo in the prostate gland, basal epithelial cells adhere to laminin 5 (LM5) via alpha3beta1 and alpha6beta4 integrins. When placed in culture primary prostate basal epithelial cells secrete and adhere to their own LM5-rich matrix. Adhesion to LM5 is required for cell survival that is dependent on integrin-mediated, ligand-independent activation of the epidermal growth factor receptor (EGFR) and the cytoplasmic tyrosine kinase Src, but not PI-3K. Integrin-mediated adhesion via alpha3beta1, but not alpha6beta4 integrin, supports cell survival through EGFR by signaling downstream to Erk. PC3 cells, which do not activate EGFR or Erk on LM5-rich matrices, are not dependent on this pathway for survival. PC3 cells are dependent on PI-3K for survival and undergo caspase-dependent death when PI-3K is inhibited. The death induced by inhibition of EGFR or Src in normal primary prostate cells is not mediated through or dependent on caspase activation, but depends on the induction of reactive oxygen species. In addition the presence of an autophagic pathway, maintained by adhesion to matrix through alpha3beta1 and alpha6beta4, prevents the induction of caspases when EGFR or Src is inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in LM5-adherent primary prostate epithelial cells.     

AMPA-mediated excitotoxicity in oligodendrocytes: role for Na(+)-K(+)-Cl(-) co-transport and reversal of Na(+)/Ca(2+) exchanger

We investigated the role of Na(+)-K(+)-Cl(-) co-transporter isoform 1 (NKCC1) and reversal of Na(+)/Ca(2+) exchanger (NCX(rev)) in glutamate-mediated excitotoxicity in oligodendrocytes obtained from rat spinal cords (postnatal day 6-8). An immunocytochemical characterization showed that these cultures express NKCC1 and Na(+)/Ca(2+) exchanger isoforms 1, 2, and 3 (NCX1, NCX2, NCX3). Exposing the cultures to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) plus cyclothiazide (CTZ) led to a transient rise in intracellular (), which was followed by a sustained overload, NKCC1 phosphorylation, and a NKCC1-mediated Na(+) influx. In the presence of a specific AMPA receptor inhibitor 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), the AMPA/CTZ failed to elicit any changes in . The AMPA/CTZ-induced sustained rise led to mitochondrial Ca(2+) accumulation, release of cytochrome c from mitochondria, and cell death. The AMPA/CTZ-elicited increase, mitochondrial damage, and cell death were significantly reduced by inhibiting NKCC1 or NCX(rev). These data suggest that in cultured oligodendrocytes, activation of AMPA receptors leads to NKCC1 phosphorylation that enhances NKCC1-mediated Na(+) influx. The latter triggers NCX(rev) and NCX(rev)-mediated overload and compromises mitochondrial function and cellular viability.     

Vasoactive intestinal peptide stimulates T lymphocytes to release IL-5 in murine schistosomiasis mansoni infection.

In murine schistosomiasis, granulomas form around ova deposited in the liver and intestines of infected mice. The granulomas have eosinophils that produce vasoactive intestinal peptide (VIP) and T cells that display VIP receptors. IL-5 is a lymphokine important for the development and maturation of eosinophils. It seemed plausible that VIP, released from eosinophils, may interact with lymphocyte VIP receptors and modulate IL-5 production as part of a feedback regulatory circuit. Thus, we determined whether granuloma T cells make IL-5 and whether VIP modulates IL-5 production. Isolated granuloma cells enriched for T lymphocytes spontaneously released IL-5. Culture of these cells in the presence of VIP increased IL-5 secretion. Spleen cells were also studied. Spleen cells from infected mice did not spontaneously release IL-5 or express IL-5 mRNA and VIP did not stimulate these resting spleen cells to produce this IL. However, these cells did express IL-5 mRNA and secreted IL-5 in response to Con A or soluble egg Ag. VIP could not appreciably modulate IL-5 release when cells were cultured with VIP and the Ag or mitogen. Spleen cells washed free of Con A ceased IL-5 secretion within 24 h. These preactivated splenic T cells resumed vigorous IL-5 secretion in response to either Con A or VIP. Yet only Con A prominently induced IL-5 mRNA expression. VIP was an effective stimulus at concentrations equal to or above the kDa of the VIP receptor on both splenic and granuloma T cells (10(-8) M). It is concluded that, in murine schistosomiasis, VIP invokes IL-5 release from activated T cells that are not undergoing immediate TCR stimulation.