The human immunodeficiency virus coat protein gp120 promotes forward trafficking and surface clustering of NMDA receptors in membrane microdomains.

Infection by the human immunodeficiency virus (HIV) can result in debilitating neurological syndromes collectively known as HIV-associated neurocognitive disorders. Although the HIV coat protein gp120 has been identified as a potent neurotoxin that enhances NMDA receptor function, the exact mechanisms for this effect are not known. Here we provide evidence that gp120 activates two separate signaling pathways that converge to enhance NMDA-evoked calcium flux by clustering NMDA receptors in modified membrane microdomains. gp120 enlarged and stabilized the structure of lipid microdomains on dendrites by mechanisms that involved a redox-regulated translocation of a sphingomyelin hydrolase (neutral sphingomyelinase-2) to the plasma membrane. A concurrent pathway was activated that accelerated the forward traffic of NMDA receptors by a PKA-dependent phosphorylation of the NR1 C-terminal serine 897 (masks an ER retention signal), followed by a PKC-dependent phosphorylation of serine 896 (important for surface expression). NMDA receptors were preferentially targeted to synapses and clustered in modified membrane microdomains. In these conditions, NMDA receptors were unable to laterally disperse and did not internalize, even in response to strong agonist induction. Focal NMDA-evoked calcium bursts were enhanced by threefold in these regions. Inhibiting membrane modification or NR1 phosphorylation prevented gp120 from accelerating the surface localization of NMDA receptors. Disrupting the structure of membrane microdomains after gp120 treatments restored the ability of NMDA receptors to disperse and internalize. These findings demonstrate that gp120 contributes to synaptic dysfunction in the setting of HIV infection by interfering with NMDA receptor trafficking.

Characteristics of progenitor cells derived from adult ciliary body in mouse, rat, and human eyes.

PURPOSE: To isolate and characterize progenitor cells derived from adult mammalian ciliary body. METHODS: The authors isolated progenitor cells from the ciliary body of adult mice, rats, and human cadaver eyes and determined quantitative growth characteristics of groups of progenitor cells called neurosphere (NS) cells, including individual cell diameter, NS diameter, percentage of NS-forming cells, and cell number per eye in mouse, rat, and human eyes. The immunolabeling and ultrastructure of NS cells were investigated by confocal and transmission electron microscopy. RESULTS: Average diameters of individual cells and neurospheres after 1 week in culture were similar in mice, rats, and humans (cell diameters: 22 +/- 1.1, 21 +/- 0.3, 25 +/- 0.4 mum; NS diameters: 139 +/- 22, 137 +/- 9, 141 +/- 11 mum, respectively). Mean numbers of cells per NS were estimated to be 1183 in mice, 5360 in rats, and 685 in humans. Molecules that were identified by immunolabeling in NS cells included nestin, Chx-10, vimentin, GFAP, and Pax-6. Thy-1 was expressed in some NS cells. Ultrastructurally, NS cells displayed abundant rough endoplasmic reticulum and many cellular processes but no characteristics of mature retinal neurons or glia. CONCLUSIONS: Progenitor cells from adult mammalian ciliary body have significant, but limited, proliferation potential and express markers characteristic of other progenitor cells and seen during early retinal development. The ciliary body could be a source of cells for transplantation in experimental rodent eyes and for autotransplantation in human eyes.

Helicobacter pylori-induced macrophage apoptosis requires activation of ornithine decarboxylase by c-Myc.

Helicobacter pylori infection causes chronic inflammation of the gastric mucosa that results from an ineffective immune response. We have demonstrated that one underlying mechanism is induction of macrophage apoptosis mediated by polyamines. The transcription factor c-Myc has been linked to induction of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. We determined whether H. pylori stimulates transcriptional activation of ODC in macrophages, whether this occurs via c-Myc, and whether these events regulate activation of apoptosis. H. pylori induced a significant increase in ODC promoter activity that peaked at 6 h after stimulation and was closely paralleled by similar increases in ODC mRNA, protein, and enzyme activity. By 2 h after stimulation, c-Myc mRNA and protein expression was induced, protein was translocated to the nucleus, and there was specific binding of a consensus probe for c-Myc to nuclear extracts. Both an antennapedia-linked inhibitor of c-Myc binding (Int-H1-S6A,F8A) and transfection of a c-Myc dominant-negative construct significantly attenuated H. pylori-induced ODC promoter activity, mRNA, enzyme activity, and apoptosis in parallel. Transfection of ODC small interfering RNA inhibited ODC activity and apoptosis to the same degree as inhibition of c-Myc binding. These studies indicate that c-Myc is an important mediator of macrophage activation and may contribute to the mucosal inflammatory response to pathogens such as H. pylori by its effect on ODC.

Spermine causes loss of innate immune response to Helicobacter pylori by inhibition of inducible nitric-oxide synthase translation.

Helicobacter pylori infection of the stomach elicits a vigorous but ineffective host immune and inflammatory response, resulting in persistence of the bacterium for the life of the host. We have reported that in macrophages, H. pylori up-regulates inducible NO synthase (iNOS) and antimicrobial NO production, but in parallel there is induction of arginase II, generating ornithine, and of ornithine decarboxylase (ODC), generating polyamines. Spermine, in particular, has been shown to restrain immune response in activated macrophages by inhibiting proinflammatory gene expression. We hypothesized that spermine could prevent the antimicrobial effects of NO by inhibiting iNOS in macrophages activated by H. pylori. Spermine did not affect the up-regulation of iNOS mRNA levels but in a concentration-dependent manner significantly attenuated iNOS protein levels and NO production. Reduction in iNOS protein was due to inhibition of iNOS translation and not due to iNOS degradation. ODC knockdown with small interfering (si) RNA resulted in increased H. pylori-stimulated iNOS protein expression and NO production without altering iNOS mRNA levels. When macrophages were cocultured with H. pylori, killing of bacteria was enhanced by transfection of ODC siRNA and prevented by addition of spermine. These results identify a mechanism of immune dysregulation induced by H. pylori in which stimulated spermine synthesis by the arginase-ODC pathway inhibits iNOS translation and NO production, leading to persistence of the bacterium and risk for peptic ulcer disease and gastric cancer.

Spermine oxidation induced by Helicobacter pylori results in apoptosis and DNA damage: implications for gastric carcinogenesis.

Oxidative stress is linked to carcinogenesis due to its ability to damage DNA. The human gastric pathogen Helicobacter pylori exerts much of its pathogenicity by inducing apoptosis and DNA damage in host gastric epithelial cells. Polyamines are abundant in epithelial cells, and when oxidized by the inducible spermine oxidase SMO(PAOh1) H(2)O(2) is generated. Here, we report that H. pylori up-regulates mRNA expression, promoter activity, and enzyme activity of SMO(PAOh1) in human gastric epithelial cells, resulting in DNA damage and apoptosis. H. pylori-induced H(2)O(2) generation and apoptosis in these cells was equally attenuated by an inhibitor of SMO(PAOh1), by catalase, and by transient transfection with small interfering RNA targeting SMO(PAOh1). Conversely, SMO(PAOh1) overexpression induced apoptosis to the same levels as caused by H. pylori. Importantly, in H. pylori-infected tissues, there was increased expression of SMO(PAOh1) in both human and mouse gastritis. Laser capture microdissection of human gastric epithelial cells demonstrated expression of SMO(PAOh1) that was significantly attenuated by H. pylori eradication. These results identify a pathway for oxidative stress-induced epithelial cell apoptosis and DNA damage due to SMO(PAOh1) activation by H. pylori that may contribute to the pathogenesis of the infection and development of gastric cancer.

Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization.

Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.