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Biomedical subjects

S A Lipton

Publications and source records attributed to S A Lipton.

At least 73 records · Page 4Linked to original sources
PubMed1998

Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice.

Intravenous tissue plasminogen activator (tPA) is used to treat acute stroke because of its thrombolytic activity and its ability to restore circulation to the brain. However, this protease also promotes neurodegeneration after intracerebral injection of excitotoxins such as glutamate, and neuronal damage after a cerebral infarct is thought to be mediated by excitotoxins. To investigate the effects of tPA on cerebral viability during ischemia/reperfusion, we occluded the middle cerebral artery in wild-type and tPA-deficient mice with an intravascular filament. This procedure allowed us to examine the role of tPA in ischemia, independent of its effect as a thrombolytic agent. tPA-deficient mice exhibited approximately 50% smaller cerebral infarcts than wild-type mice. Intravenous injection of tPA into tPA-/- or wild-type mice produced larger infarcts, indicating that tPA can increase stroke-induced injury. Since tPA promotes desirable (thrombolytic) as well as undesirable (neurotoxic) outcomes during stroke, future therapies should be aimed at countering the excitotoxic damage of tPA to afford even better neuroprotection after an acute cerebral infarct.

Animals↗
PubMed1998

Role of caspases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons.

Overactivation of glutamate receptors mediates neuronal death in several acute and chronic neurodegenerative diseases. The intracellular processes underlying this form of death, however, remain poorly understood. Depending on the severity of insult, N-methyl-D-aspartate (NMDA) receptor activation induces either apoptosis or necrosis. Cysteine proteases related to interleukin-1beta-converting enzyme (ICE), recently termed caspases, appear necessary for neuronal apoptosis in vivo and in vitro. To determine whether caspases play a role in NMDA-induced apoptosis, we used two functionally distinct approaches to decrease substrate cleavage by caspases. One is a novel peptide (V-ICEinh) that contains the caspase catalytic site and acts as a pseudoenzyme that binds caspase substrates and prevents their cleavage. The other is a pseudosubstrate peptide (Z-VAD x fmk) that inhibits caspase activity. Pretreatment with either V-ICEinh or Z-VAD-fmk protects cerebrocortical neurons from NMDA-induced apoptosis, suggesting a role for caspases in NMDA-induced apoptosis. To explore the signaling pathways involved, we looked at the effects of NMDA receptor activation on Ca2+ influx, production of reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation. Neither NMDA-induced Ca2+ influx nor the initial collapse of mitochondrial membrane potential could be prevented by pretreatment with V-ICEinh or Z-VAD x fmk. In contrast, ROS formation and lipid peroxidation were completely blocked by both V-ICEinh and Z-VAD x fmk. Taken together, our results suggest that Ca2+ influx and mitochondrial depolarization occur upstream from caspase activation, whereas ROS formation and lipid peroxidation may be downstream events in the cascade leading to cortical neuronal apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Neuronal injury associated with HIV-1: approaches to treatment.

Mounting evidence suggests that cognitive dysfunction developing as a result of HIV-1 infection is mediated at least in part by generation of excitotoxins and free radicals in the brain. This syndrome is currently designated HIV-1-associated cognitive/motor complex, was originally termed the AIDS Dementia Complex, and for simplicity, is called AIDS dementia in this review. Recently, brains of patients with AIDS have been shown to manifest neuronal injury and 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 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 and microglia, or immune-activated macrophages and astrocytes (activated by the shed HIV-1 envelope protein, gp120, or other viral proteins and cytokines), appear to secrete excitants and neurotoxins. These substances may include arachidonic acid, platelet-activating factor, free radicals (NO. and O2.-), glutamate, quinolinate, cysteine, amines, and as yet unidentified factors emanating from stimulated macrophages and reactive astrocytes. A final common pathway for neuronal susceptibility is 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, there is hope for future pharmacological intervention.

AIDS Dementia Complex↗
PubMed1998

A phase I/II trial of nimodipine for HIV-related neurologic complications.

BACKGROUND: Few effective treatments are available for AIDS dementia complex (ADC) and HIV-associated neuropathy. However, recent in vitro studies indicate that nimodipine, a voltage-dependent calcium channel antagonist, can prevent HIV-related neuronal injury and may provide a novel form of treatment for these disorders. METHODS: To determine the safety and possible efficacy of this agent, 41 patients with mild to severe ADC, including 19 patients with neuropathy, were entered into the AIDS Clinical Trial Group multicenter, phase-I and phase-II study. Nimodipine at 60 mg p.o., five times daily; 30 mg p.o., three times daily; or placebo was administered for 16 weeks as adjuvant treatment to antiretroviral therapy. RESULTS: Neuropsychological performance at baseline, measured by the composite neuropsychological Z score (NPZ-8), correlated significantly with the ADC stage and with CSF levels of neopterin, a marker of immune activation. No significant differences in toxicity were observed among the three arms. Intent-to-treat analysis showed no significant change in the NPZ-8, although improvement was suggested in the high-dose arm. In addition, a trend toward stabilization in peripheral neuropathy was observed in both nimodipine arms compared with placebo. CONCLUSIONS: Nimodipine and other similar nonantiretroviral agents may provide a safe and promising avenue of treatment for neurologic disorders associated with HIV infection. The results of this study indicate that further clinical trials are warranted.

AIDS Dementia Complex↗
PubMed1997

Enhanced neuronal death from focal ischemia in AMPA-receptor transgenic mice.

Excitatory amino acid (EAA) receptors play an important role in neuronal cell death in acute cerebral ischemia. Blocking the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of EAA receptor has been shown to reduce cell death in global cerebral ischemia. However their role in focal stroke, although suggestive, has remained more contentious. To clarify this issue, we generated transgenic mice overexpressing the AMPA receptor (AMPAR) subunit GluR2-flip which would increase AMPAR-mediated currents. Excitatory neurons in these transgenic mice are thus predicted to be more susceptible than wild-type neurons to EAA (glutamate)-induced excitotoxic damage. Consistent with this prediction, cultured neurons from transgenic mice had a lower LD50 for exposure to glutamate (10(-3)-10(-5) M for 5 min) compared to wild-type neurons. Moreover, transgenic mice subjected to permanent focal ischemia of the middle cerebral artery (MCA) using the intralumenal filament model sustained larger infarctions compared to wild-type controls. Hence we have developed a genetic mouse model that demonstrates the crucial role of AMPAR containing GluR2-flip in the pathogenesis of focal hypoxic-ischemic neuronal cell death. This model will be a valuable tool in elucidating molecular mechanisms of glutamate excitotoxicity and evaluating the efficacy of glutamate receptor antagonists in attenuating post-ischemic neuronal cell death.

Alternative Splicing↗
PubMed1997

Suppression of neuronal apoptosis by S-nitrosylation of caspases.

S-Nitrosylation (reaction of nitric oxide (NO) species with a critical cysteine sulfhydryl) can regulate the physiological activity of proteins, including enzymes, ion channels, G-proteins, and transcription factors. Caspases are a family of interleukin-1beta-converting enzyme-like proteases involved in the signaling pathway to apoptotic cell death, and each member of this enzyme family contains a critical cysteine residue in its active site. Here we show that S-nitrosylation of caspases in human embryonic kidney (HEK)-293 cells and primary cerebrocortical neurons decreases enzyme activity and is associated with protection from apoptosis.

Apoptosis↗
PubMed1997

Agonist-induced closure of constitutively open gamma-aminobutyric acid channels with mutated M2 domains.

Ligand-gated ion channels display a fundamental property-channels remain virtually closed at rest and open upon agonist binding. Here we show that substituting alanines for either of two amino acid residues (T314 or L317) in the M2 region of the gamma-aminobutyric acid (GABA) rho1 subunit results in spontaneous channel opening in the absence of ligand. Surprisingly, for two single point mutants (T314A or L317A), application of very low concentrations of agonist partially suppressed this spontaneous current, while higher concentrations re-activated the receptors. When both of these sites were mutated (T314A/L317A), GABA nearly completely suppressed the constitutive current and did not re-activate the current even at very high concentrations. This study provides important new insights into the structure-function relationship of ligand-gated ion channels, where modification of the structure of the channel pore region not only alters the allosteric transition of the receptor protein but also reverses the polarity of agonist regulation of channel gating. Our results suggest that the sites where these two residues are located are structurally critical for channel gating.

Amino Acid Sequence↗
PubMed1997

Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor.

Severely elevated levels of total homocysteine (approximately millimolar) in the blood typify the childhood disease homocystinuria, whereas modest levels (tens of micromolar) are commonly found in adults who are at increased risk for vascular disease and stroke. Activation of the coagulation system and adverse effects of homocysteine on the endothelium and vessel wall are believed to underlie disease pathogenesis. Here we show that homocysteine acts as an agonist at the glutamate binding site of the N-methyl-D-aspartate receptor, but also as a partial antagonist of the glycine coagonist site. With physiological levels of glycine, neurotoxic concentrations of homocysteine are on the order of millimolar. However, under pathological conditions in which glycine levels in the nervous system are elevated, such as stroke and head trauma, homocysteine's neurotoxic (agonist) attributes at 10-100 microM levels outweigh its neuroprotective (antagonist) activity. Under these conditions neuronal damage derives from excessive Ca2+ influx and reactive oxygen generation. Accordingly, homocysteine neurotoxicity through overstimulation of N-methyl-D-aspartate receptors may contribute to the pathogenesis of both homocystinuria and modest hyperhomocysteinemia.

2-Amino-5-phosphonovalerate↗
PubMed1997

Mechanism of memantine block of NMDA-activated channels in rat retinal ganglion cells: uncompetitive antagonism.

1. N-methyl-D-aspartic acid (NMDA)-activated currents were recorded from dissociated rat retinal ganglion cells using whole-cell recording. The NMDA open-channel blocking drug memantine was evaluated for non-competitive and/or uncompetitive components of antagonism. A rapid superfusion system was used to apply various drugs for kinetic analysis. 2. Dose-response data revealed that memantine blocked 200 microM NMDA-evoked responses with a 50% inhibition constant (IC50) of approximately 1 microM at -60 mV and an empirical Hill coefficient of approximately 1. The antagonism followed a bimolecular reaction process. This 1:1 stoichiometry is supported by the fact that the macroscopic blocking rate of memantine (kon) increased linearly with memantine concentration and the macroscopic unblocking rate (koff) was independent of it. The estimated pseudo-first order rate constant for macroscopic blockade was 4 x 10(5) M-1 S-1 and the rate constant for unblocking was 0.44 s-1. Both the blocking and unblocking actions of memantine were well fitted by a single exponential process. 3. The kon for 2 microM memantine decreased with decreasing concentrations of NMDA. By analysing kon behaviour, we estimate that memantine has minimal interaction with the closed-unliganded state of the channel. As channel open probability (Po) approached zero, a small residual action of memantine may be explained by the presence of endogenous glutamate and glycine. 4. Memantine could be trapped within the NMDA-gated channel if it was suddenly closed by fast washout of agonist. The measured gating process of channel activation and deactivation appeared at least 10-20-fold faster than the kinetics of memantine action. By combining the agonist and voltage dependence of antagonism, a trapping scheme was established for further kinetic analysis. 5. With low agonist concentrations, NMDA-gated channels recovered slowly from memantine blockade. By analysing the probability of a channel remaining blocked, we found that memantine binding appeared to stabilize the open conformation of the blocked channel and did not affect ligand affinity. Validity of the "trapping model' and stabilization of the open conformation were further suggested by agreement between the predicted dose-response curve for NMDA in the presence of 2 microM memantine and the empirically derived dose-response relationship. 6. Based on simple molecular schemes, the degree of blockade at various concentrations of agonist for "pure' non-competitive vs. uncompetitive inhibition was computer simulated. The measured degree of blockade by 6 microM memantine was close to ideal for pure uncompetitive antagonism. Taken together, we conclude that the predominant mechanism of open-channel blockade by memantine is uncompetitive. In general, the relative magnitude of the dissociation rate of an open-channel blocker from the open but blocked channel (the apparent off-rate) compared with the rate of leaving the closed and blocker-trapped state (the leak rate) will determine the contribution of uncompetitive vs. non-competitive actions, respectively. 7. Millimolar internal Cs+ competed with memantine for binding in the NMDA-gated channel, and reduced the association rate of memantine, but had no effect on the voltage dependence of the dissociation rate. After removal of Cs+, the calculated Ki for memantine remained voltage dependent. These observations would be difficult to reconcile with models in which memantine binds to a site outside the channel pore and instead strongly support the supposition that the blocking site for memantine is within the permeation pathway.

Animals↗
PubMed1997

Molecular basis of glutamate toxicity in retinal ganglion cells.

Loss of retinal ganglion cells (RGCs) is a hallmark of many ophthalmic diseases including glaucoma, retinal ischemia due to central artery occlusion, anterior ischemic optic neuropathy and may be significant in optic neuritis, optic nerve trauma, and AIDS. Recent research indicates that neurotoxicity is caused by excessive stimulation of receptors for excitatory amino acids (EAAs). In particular, the amino acid glutamate has been shown to act as a neurotoxin which exerts its toxic effect on RGCs predominantly through the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. NMDA-receptor-mediated toxicity in RGCs is dependent on the influx of extracellular Ca2+. The increase in [Ca2+]i acts as a second messenger that sets in motion the cascade leading to eventual cell death. Glutamate stimulates its own release in a positive feedback loop by its interaction with the non-NMDA receptor subtypes. Ca(2+)-induced Ca2+ release and further influx of Ca2+ through voltage-gated Ca2+ channels after glutamate-induced depolarization contribute to glutamate toxicity. In vitro and in vivo studies suggest that the use of selective NMDA receptor antagonists or Ca2+ channel blockers should be useful in preventing or at least abating neuronal loss in the retina. Of particular importance for future clinical use of NMDA receptor antagonists in the treatment of acute vascular insults is the finding that some drugs can prevent glutamate-induced neurotoxicity, even when administered a few hours after the onset of retinal ischemia.

Animals↗
PubMed1997

Neuropathogenesis of acquired immunodeficiency syndrome dementia.

During the past year progress has been made in our understanding of the pathogenesis of the dementia associated with the acquired immunodeficiency syndrome. As many as one-third of acquired immunodeficiency syndrome patients eventually develop this condition, and at present it remains only poorly or transiently treated by existing antiretroviral therapies which do not penetrate well into the central nervous system. The past year has witnessed further characterization of microglial/macrophage neurotoxins, increasing evidence for neuronal death by apoptosis, and a more quantitative search for viral products, surrogate markers, or magnetic resonance spectroscopic parameters of brain or cerebrospinal fluid, or both. An increased understanding that the mediation of neuronal injury is not by direct infection of neurons, but rather via a complex network of cytokines, excitotoxins, and free radical mechanisms triggered by human immunodeficiency virus-infected or immune-stimulated brain macrophages and astrocytes has led to the development of therapies that are administered adjunctively with antiretroviral drugs. Some of these potential new treatments have now entered clinical trials.

AIDS Dementia Complex↗
PubMed1996

Nitric oxide-related species inhibit evoked neurotransmission but enhance spontaneous miniature synaptic currents in central neuronal cultures.

Nitric oxide (NO.) does not react significantly with thiol groups under physiological conditions, whereas a variety of endogenous NO donor molecules facilitate rapid transfer to thiol of nitrosonium ion (NO+, with one less electron than NO.). Here, nitrosonium donors are shown to decrease the efficacy of evoked neurotransmission while increasing the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs). In contrast, pure NO donors have little effect (displaying at most only a slight increase) on the amplitude of evoked EPSCs and frequency of spontaneous mEPSCs in our preparations. These findings may help explain heretofore paradoxical observations that the NO moiety can either increase, decrease, or have no net effect on synaptic activity in various preparations.

Animals↗
PubMed1996

BCL-2 delay apoptosis and PARP cleavage induced by NO donors in GT1-7 cells.

BCL-2 is a negative regulator of cell death in several systems. Here we report that bcl-2 expression protects against apoptosis induced by nitric oxide (NO) donors in GT1-7 hypothalamic cells. BCL-2 significantly inhibited neuronal death caused by 200 microM S-nitroso-cysteine (SNOC), 200 microM S-nitroso-N-acetyl-penicillamine (SNAP), or 1 mM 3-morpholinosydnonimine (SIN-1). To explore further the protective mechanism(s) elicited by bcl-2 expression, we investigated whether BCL-2 could prevent NO-induced cleavage of poly-ADP-ribose-polymerase (PARP), which is a substrate for interleukin-1 beta converting enzyme (ICE)-like proteases in apoptosis. Formation of 85 and 25 kDa PARP fragments elicited by NO donors was inhibited in cells over-expressing bcl-2.

Apoptosis↗
PubMed1996

Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma.

OBJECTIVE: To explore the possibility that the excitatory amino acid glutamate might be associated with the disease process of glaucoma, which is characterized by the death of retinal ganglion cell neurons and subsequent visual dysfunction. METHODS: Amino acid analyses were performed on vitreous specimens that were obtained from patients who were undergoing cataract extraction. Samples were collected prospectively from those patients who sustained inadvertent rupture of the posterior capsule between 1988 and 1993. An additional set of specimens, obtained from both eyes of monkeys, was analyzed; in these monkeys, glaucoma had been experimentally induced in one eye only. RESULTS: A twofold elevation in the level of glutamate was detected in the vitreous body of the group of patients with glaucoma when compared with that in a control population of patients with cataracts only. An even greater elevation of the glutamate level was found in the vitreous body of glaucomatous eyes of monkeys when compared with that in control eyes. No statistical differences were detected among other amino acid levels from the vitreous body of glaucomatous and nonglaucomatous eyes in humans or monkeys. CONCLUSIONS: The excitatory amino acid glutamate is found in the vitreous body of glaucomatous eyes at concentrations that are potentially toxic to retinal ganglion cells. The increased level of this known neurotoxin is consistent with an "excitotoxic" mechanism for the retinal ganglion cell and optic nerve damage in glaucoma. Therapies to protect neurons against glutamate toxic effects may prove to be useful in the management of this blinding disease.

Aged↗