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Biomedical subjects

S A Lipton

Publications and source records attributed to S A Lipton.

At least 91 records · Page 5Linked to original sources
PubMed1996

Intercellular adhesion molecule-1-deficient mice are less susceptible to cerebral ischemia-reperfusion injury.

Neutrophil emigration is mediated by adhesion proteins that are highly expressed on the endothelial surface during inflammatory processes in the brain. Intercellular adhesion molecule-1 (ICAM-1) is an inducible adhesion molecule that binds to leukocyte integrins and facilitates neutrophil adhesion and transendothelial migration. To study the role of ICAM-1 during ischemia and reperfusion in the brain, we analyzed the effect of transient focal cerebral ischemia in ICAM-1-deficient mice generated by gene targeting in embryonic stem cells. Transient focal ischemia was induced by occluding the left middle cerebral artery for 3 hours followed by a 21- or 45-hour reperfusion period. When compared with their wild-type littermates, ICAM-1-deficient mice were less susceptible to cerebral injury as demonstrated by a 5.6- or 7.8-fold reduction in infarction volume, respectively. These data support the premise that neutrophil adhesion in ischemic areas may be deleterious and that ICAM-1 deficiency reduces neurological damage after transient focal cerebral ischemia.

Animals↗
PubMed1996

Cytoskeletal breakdown and apoptosis elicited by NO donors in cerebellar granule cells require NMDA receptor activation.

We have recently demonstrated that nitric oxide (NO) donors can trigger either apoptosis or necrosis of neurons as a function of the intensity of the exposure. Here, we show that the apoptosis induced by the NO donors S-nitrosocysteine (SNOC) or S-nitroso-N-acetyl-penicillamine (SNAP) in cultured cerebellar granule cells (CGCs) depends on NMDA receptor (NMDA-R) activation leading to intracellular Ca2+ overload. Early dissolution of actin filaments followed by breakdown of microtubules and nuclear lamins preceded the appearance of typical apoptotic features. NO donors induced tyrosine nitration in neurons, in a small population of contaminating astrocytes, and in cultures of cerebellar astroglial cells. However, astrocytes neither displayed cytoskeletal alterations nor underwent apoptosis. Competitive and uncompetitive NMDA receptor antagonists, such as D-aminophosphonovaleric acid and MK-801, did not influence tyrosine nitration but prevented the accumulation of intracellular Ca2+, cytoskeletal breakdown, and apoptosis induced by either SNOC or SNAP in CGCs. Taken together, these data strongly suggest that Ca2+ influx through NMDA-R-gated ion channels is a critical event in CGC apoptosis induced by NO donors.

Animals↗
PubMed1996

Similarity of neuronal cell injury and death in AIDS dementia and focal cerebral ischemia: potential treatment with NMDA open-channel blockers and nitric oxide-related species.

Using in vitro models, our laboratory in collaboration with those of Pierluigi Nicotera (University of Konstanz, Germany) and Stan Orrenius (Karolinska Institute) has recently shown that fulminant insults to the nervous system from excitotoxins or free radicals result in neuronal cell death from necrosis, while more subtle insults result in delayed apoptosis. Over the past dozen or so years, mounting evidence has suggested that excitotoxins, such as glutamate, result in neuronal cell death after stroke. More recent evidence has suggested that in addition to necrotic cell death in the ischemic core, a number of neurons may also undergo apoptosis. Thus, the hypothesis that intense injury leads to necrosis while mild insult (perhaps in the penumbra) leads to apoptosis may hold in focal cerebral ischemia. Another neurological malady with mounting evidence for a pathogenesis that is mediated at least in part by excitotoxins is HIV-1-associated cognitive/motor complex (originally termed the AIDS Dementia Complex and, for convenience, designated here AIDS dementia). AIDS dementia appears to be associated with several neuropathological abnormalities, including giant cell formation by microglia, astrogliosis, and neuronal injury or loss. Recently, neuronal and other cell injury in AIDS brains has been shown to result in apoptotic-like cell death. How can HIV-1 result in neuronal damage if neurons themselves are only rarely, if ever, infected by the virus? Experiments from several different laboratories, including our group in collaboration with that of Howard Gendelman (University of Nebraska Medical Center), have lent support to the existence of HIV- and immune-related toxins in a variety of in vitro and in vivo paradigms. In one recently defined pathway to neuronal injury, HIV-infected macrophages/ microglia as well as macrophages activated by HIV-1 envelope protein gp120 appear to secrete excitants/ neurotoxins. These substances may include arachidonic acid, platelet-activating factor, free radicals (NO. and O2.-), glutamate, quinolinate, cysteine, cytokines (TNF-alpha, IL1-beta, IL-6), amines, and as yet unidentified factors emanating from stimulated macrophages and possibly reactive astrocytes. A final common pathway for neuronal susceptibility appears to be operative, similar to that observed in stroke and several neurodegenerative diseases. This mechanism involves excessive activation of N-methyl-D-aspartate (NMDA) receptor-operated channels, with resultant excessive influx of Ca2+ and the generation of free radicals, leading to neuronal damage. With the very recent development of clinically-tolerated NMDA antagonists, as discussed here, there is hope for future pharmacological intervention.

AIDS Dementia Complex↗
PubMed1996

Chronic low-dose glutamate is toxic to retinal ganglion cells. Toxicity blocked by memantine.

PURPOSE: It is well known that acute exposure to high concentrations of glutamate is toxic to central mammalian neurons. However, the effect of a chronic, minor elevation over endogenous glutamate levels has not been explored. The authors have suggested that such chronic exposure may play a role in glaucomatous neuronal loss. In the current study, they sought to explore whether a chronic, low-dose elevation in vitreal glutamate was toxic to retinal ganglion cells and whether this toxicity could be prevented with memantine, a glutamate antagonist. METHODS: Rats were injected serially and intravitreally with glutamate to induce chronic elevations in glutamate concentration. A second group of rats was treated with intraperitoneal memantine and glutamate. Control groups received vehicle injection with or without concurrent memantine therapy. After 3 months, the animals were killed, and ganglion cell survival was evaluated. RESULTS: Intravitreal injections raised the intravitreal glutamate levels from an endogenous range of 5 to 12 microM glutamate to 26 to 34 microM. This chronic glutamate elevation killed 42% of the retinal ganglion cells after 3 months. Memantine treatment alone had no effect on ganglion cell survival. However, when memantine was given concurrently with low-dose glutamate, memantine was partially protective against glutamate toxicity. CONCLUSIONS: These data suggest that minor elevations in glutamate concentration can be toxic to ganglion cells if this elevation is maintained for 3 months. Furthermore, memantine is efficacious at protecting ganglion cells from chronic low-dose glutamate toxicity.

Animals↗
PubMed1996

NMDA receptors: from genes to channels.

N-methyl-D-aspartate receptors belong to the family of ionotropic glutamate receptors. NMDA receptors were named after the specific glutamate-like synthetic agonist N-methyl-D-aspartate. In the past decade, an increasing number of functional sites have been discovered and used to refine the operational definition of NMDA receptors. The goal to characterize the molecular substrate underlying the heretofore strictly operationally defined NMDA receptors has come into reach following the cloning of a number of cDNAs coding for NMDA receptor subunits. However, in their review, Nikolaus Sucher and colleagues show that caution should be exercised in comparing the pharmacological properties of recombinant NMDA receptors to those of native neurones. Future work on NMDA receptors will be challenged to reconcile disparate effects obtained with recombinant versus native receptors.

Humans↗
PubMed1995

Cloning of a gamma-aminobutyric acid type C receptor subunit in rat retina with a methionine residue critical for picrotoxinin channel block.

Ionotropic receptors for gamma-aminobutyric acid (GABA) are important to inhibitory neurotransmission in the mammalian retina, mediating GABAA and GABAC responses. In many species, these responses are blocked by the convulsant picrotoxinin (PTX), although the mechanism of block is not fully understood. In contrast, GABAC responses in the rat retina are extremely resistant to PTX. We hypothesized that this difference could be explained by molecular characterization of the receptors underlying the GABAC response. Here we report the cloning of two rat GABA receptor subunits, designated r rho 1 and r rho 2 after their previously identified human homologues. When coexpressed in Xenopus oocytes, r rho 1/r rho 2 heteromeric receptors mimicked PTX-resistant GABAC responses of the rat retina. PTX resistance is apparently conferred in native heteromeric receptors by r rho 2 subunits since homomeric r rho 1 receptors were sensitive to PTX; r rho 2 subunits alone were unable to form functional homomeric receptors. Site-directed mutagenesis confirmed that a single amino acid residue in the second membrane-spanning region (a methionine in r rho 2 in place of a threonine in r rho 1) is the predominant determinant of PTX resistance in the rat receptor. This study reveals not only the molecular mechanism underlying PTX blockade of GABA receptors but also the heteromeric nature of native receptors in the rat retina that underlie the PTX-resistant GABAC response.

Amino Acid Sequence↗
PubMed1995

The coat protein gp120 of HIV-1 inhibits astrocyte uptake of excitatory amino acids via macrophage arachidonic acid.

The human immunodeficiency virus coat protein gp120 injures central mammalian neurons both in vitro and in vivo, and this observation may contribute, at least in part, to the neurological dysfunction associated with the acquired immunodeficiency syndrome. Recent work suggests that gp120 mediates neuronal damage predominantly via an indirect route involving activation of brain macrophages. We have previously shown that the stimulation of N-methyl-D-aspartate receptors by excitatory amino acids is essential for the neuronal injury observed with gp120. Here we show that gp120 impairs astrocyte uptake of excitatory amino acids and the excess glutamate thus engendered may contribute to the increased neuronal damage. We also studied the mechanism whereby gp120 inhibits the uptake of excitatory amino acids by astrocytes. We present data suggesting that at least one pathway involves a direct effect of gp120 on macrophages, which in turn release arachidonic acid, a known inhibitor of excitatory amino acid uptake by astrocytes. Our findings suggest that the observed effects on glia and neurons of gp120 may be secondary, at least in part, to its initial activation of macrophages.

Acquired Immunodeficiency Syndrome↗
PubMed1995

An astrocytic binding site for neuronal Thy-1 and its effect on neurite outgrowth.

Thy-1, a member of the immunoglobulin superfamily, is one of the most abundant glycoproteins on mammalian neurons. Nevertheless, its role in the peripheral or central nervous system is poorly understood. Certain monoclonal antibodies to Thy-1 promote neurite outgrowth by rodent central nervous system neurons in vitro, suggesting that Thy-1 functions, in part, by modulating neurite outgrowth. We describe a binding site for Thy-1 on astrocytes. This Thy-1-binding protein has been characterized by immunofluroesence with specific anti-idiotype monoclonal antibodies and by three competitive binding assays using (i) anti-idiotype antibodies, (ii) purified Thy-1, and (iii) Thy-1-transfected cells. The Thy-1-binding protein may participate in axonal or dendritic development in the nervous system.

Animals↗
PubMed1995

Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures.

N-Methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity may depend, in part, on the generation of nitric oxide (NO.) and superoxide anion (O2.-), which react to form peroxynitrite (OONO-). This form of neurotoxicity is thought to contribute to a final common pathway of injury in a wide variety of acute and chronic neurologic disorders, including focal ischemia, trauma, epilepsy, Huntington disease, Alzheimer disease, amyotrophic lateral scelerosis, AIDS dementia, and other neurodegenerative diseases. Here, we report that exposure of cortical neurons to relatively short durations or low concentrations of NMDA, S-nitrosocysteine, or 3-morpholinosydnonimine, which generate low levels of peroxynitrite, induces a delayed form of neurotoxicity predominated by apoptotic features. Pretreatment with superoxide dismutase and catalase to scavenge O2.- partially prevents the apoptotic process triggered by S-nitrosocysteine or 3-morpholinosydnonimine. In contrast, intense exposure to high concentrations of NMDA or peroxynitrite induces necrotic cell damage characterized by acute swelling and lysis, which cannot be ameliorated by superoxide dismutase and catalase. Thus, depending on the intensity of the initial insult, NMDA or nitric oxide/superoxide can result in either apoptotic or necrotic neuronal cell damage.

Animals↗
PubMed1995

Transcriptional or translational inhibition blocks low dose NMDA-mediated cell death.

Glutamate toxicity in nerve cells has been well documented and may play a role in a broad spectrum of neurological and ophthalmic diseases. Recent work in several laboratories has suggested that an apoptotic-like mechanism may be implicated in glutamate toxicity under certain circumstances. We therefore studied the effects of transcriptional and translational inhibition on glutamate-mediated cell death in retinal ganglion cells. We now report that either cycloheximide or actinomycin D can, even when added 2 h after the initial excitotoxic insult, save retinal ganglion cells from low dose glutamate toxicity. However, cycloheximide or actinomycin D are unable to prevent glutamate-mediated death at higher concentrations of excitotoxin. This result indicates that at low doses, the neurotoxic effects of glutamate may develop through an apoptotic-like mechanism.

Animals↗
PubMed1995

Regulation of nitric oxide synthase activity in human immunodeficiency virus type 1 (HIV-1)-infected monocytes: implications for HIV-associated neurological disease.

Mononuclear phagocytes (monocytes, macrophages, and dendritic cells) play major roles in human immunodeficiency virus (HIV) persistence and disease pathogenesis. Macrophage antigen presentation and effector cell functions are impaired by HIV-1 infection. Abnormalities of macrophage effector cell function in bone marrow, lung, and brain likely result as a direct consequence of cellular activation and HIV replication. To further elucidate the extent of macrophage dysfunction in HIV-1 disease, a critical activation-specific regulatory molecule, nitric oxide (NO.), which may contribute to diverse pathology, was studied. Little, if any, NO. is produced by uninfected human monocytes. In contrast, infection with HIV-1 increases NO. production to modest, but significant levels (2-5 microM). Monocyte activation (with lipopolysaccharide, tumor necrosis factor alpha, or through interactions with astroglial cells) further enhances NO. production in HIV-infected cells, whereas its levels are diminished by interleukin 4. These results suggest a possible role for NO. in HIV-associated pathology where virus-infected macrophages are found. In support of this hypothesis, RNA encoding the inducible NO synthase (iNOS) was detected in postmortem brain tissue from one pediatric AIDS patient with advanced HIV encephalitis. Corresponding iNOS mRNA was not detected in brain tissue from five AIDS patients who died with less significant brain disease. These results demonstrate that HIV-1 can influence the expression of NOS in both cultured human monocytes and brain tissue. This newly described feature of HIV-macrophage interactions suggests previously unappreciated mechanisms of tissue pathology that result from productive viral replication.

Amino Acid Oxidoreductases↗
PubMed1995

N-methyl-D-aspartate receptors are critical for mediating the effects of glutamate on intracellular calcium concentration and immediate early gene expression in cultured hippocampal neurons.

The mechanisms by which activation of excitatory amino acid receptors is coupled to the regulation of gene transcription were studied using cultured hippocampal neurons from neonatal rats. Voltage recording, calcium imaging, specific RNA analysis and immunocytochemistry were carried out on sister cultures. This allowed analysis of the expression of functional glutamate receptor subtypes, examination of their role in controlling intracellular free calcium ([Ca2+]i), and determination of their relative contributions to the transcriptional regulation of six immediate early genes c-fos, fosB, c-jun, junB, zif/268 (also termed Egr-1; NGFI-A; Krox-24) and nur/77 (also termed NGFI-B). Expression of all six immediate early genes was induced in hippocampal neurons by glutamate treatment. Nuclear run-on assays demonstrated that this induction occurred at the transcriptional level. Activation of the N-methyl-D-aspartate subtype of glutamate receptor was necessary and sufficient for the transcriptional response. Non-N-methyl-D-aspartate receptors, while present in cultured hippocampal neurons, contributed relatively little to the regulation of transcription. Calcium imaging showed that glutamate-induced changes in [Ca2+]i were almost entirely mediated by N-methyl-D-aspartate receptors, rather than by L-type voltage-sensitive calcium channels. Previous studies have shown that stimulation with selective agonists of either N-methyl-D-aspartate receptors, non-N-methyl-D-aspartate receptors, or L-type calcium channels can lead to an increase in [Ca2+]i and c-fos expression. Here we demonstrate that in our hippocampal culture system glutamate controls [Ca2+]i and induces immediate early gene transcription primarily by activating N-methyl-D-aspartate receptors.

2-Amino-5-phosphonovalerate↗
PubMed1995

Co-expression of AMPA/kainate receptor-operated channels with high and low Ca2+ permeability in single rat retinal ganglion cells.

The patch-clamp technique was used to record whole-cell currents induced by alpha-amino-3-hydroxyl-5-methyl-isoxazol-4-propionic acid (AMPA) or kainate in solitary rat retinal ganglion cells (n = 125) in vitro. Two groups of retinal ganglion cells could be distinguished according to their responses to kainate or AMPA in extracellular solutions with Ca2+ as the only permeant cation. The ratio of the steady-state currents evoked by a given concentration of AMPA compared to kainate was low (0.08) in the first group and high (0.61) in the second group of retinal ganglion cells. The Ca2+ permeability through AMPA/kainate receptor-operated channels was low (PCa2+/PCs+ < 0.1) in the first group (n = 74, 59%) and moderate (PCa2+/PCs+ = 0.53) in the second group (n = 51, 41%) of retinal ganglion cells. The fraction of the total current induced by stimulation of non-N-methyl-D-aspartate receptors that is flowing through Ca2+ permeable AMPA/kainate channels in single cells with high Ca2+ permeability was estimated by comparing the current-voltage relationship in extracellular solutions with either Ca2+ or Na+ as the sole charge carrier. The contribution of Ca(2+)-permeable channels to the non-N-methyl-D-aspartate receptor induced whole-cell current in single Ca2+ permeable cells (n = 12) ranged from 40 to 70%, correlating with the intermediate level of Ca2+ permeability (PCa2+/PCs+ = 0.22-0.80) measured by an independent method in these cells. Thus, single Ca(2+)-permeable cells appear to express at least two types of AMPA/kainate receptor-operated channels with high or low Ca2+ permeability. Using the polymerase chain reaction, transcripts for the glutamate receptor subunits 1-4, including their "flip" and "flop" versions, were identified in retinal ganglion cells. Together, these findings suggest that among rat retinal ganglion cells there are differences in the pattern of expression of AMPA/kainate receptor-operated channels. Moreover, individual cells co-express multiple heterologous non-N-methyl-D-aspartate receptors with distinct functional properties. The functional diversity of these receptors may play an important role in controlling Ca2+ entry into neurons. We speculate that the low Ca2+ permeability and the preference for kainate in one group of retinal ganglion cells may be due to the predominant expression of non-N-methyl-D-aspartate receptors containing the edited form of the glutamate receptor subunit 2 flop splice variant.

Animals↗