Biomedical subjects
S A Lipton
Publications and source records attributed to S A Lipton.
Expression of endogenous NMDAR1 transcripts without receptor protein suggests post-transcriptional control in PC12 cells.
Expression of RNA for the NMDAR1 subunit of the N-methyl-D-aspartate receptor was detected by Northern hybridization in both nerve growth factor-differentiated and undifferentiated rat pheochromocytoma (PC12) cells. The NMDA receptor type 1 (NMDAR1) message in PC12 cells was similar in size to that expressed in hippocampal neurons. PC12 cell cDNAs that were amplified by polymerase chain reaction with primers flanking the coding region of NMDAR1 corresponded to the NMDAR1 splice variant NMDA receptor type 1 isoform C (NMDAR1C). Using calcium imaging or patch-clamp recording, no functional NMDA-gated ion channels were found in PC12 cells. A monoclonal antibody against NMDAR1 was developed in order to investigate whether or not NMDAR1 protein was present in PC12 cells. Only trace amounts of NMDAR1 protein were found in native PC12 cells. However, expression of NMDAR1 protein was detected in PC12 cells that were transfected with an expression vector containing an NMDAR1C clone under control of a cytomegalovirus promoter. These findings suggest that the expression of NMDAR1 protein in PC12 cells may be controlled by post-transcriptional mechanisms. The PC12 cell line may serve as a model system for the study of the transcriptional, post-transcriptional, and translational regulation of NMDAR1. Furthermore, the presence of NMDAR1 RNA in a particular cell type may not necessarily indicate expression of NMDAR1 protein.
A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds.
Congeners of nitrogen monoxide (NO) are neuroprotective and neurodestructive. To address this apparent paradox, we considered the effects on neurons of compounds characterized by alternative redox states of NO: nitric oxide (NO.) and nitrosonium ion (NO+). Nitric oxide, generated from NO. donors or synthesized endogenously after NMDA (N-methyl-D-aspartate) receptor activation, can lead to neurotoxicity. Here, we report that NO.- mediated neurotoxicity is engendered, at least in part, by reaction with superoxide anion (O2.-), apparently leading to formation of peroxynitrite (ONOO-), and not by NO. alone. In contrast, the neuroprotective effects of NO result from downregulation of NMDA-receptor activity by reaction with thiol group(s) of the receptor's redox modulatory site. This reaction is not mediated by NO. itself, but occurs under conditions supporting S-nitrosylation of NMDA receptor thiol (reaction or transfer of NO+). Moreover, the redox versatility of NO allows for its interconversion from neuroprotective to neurotoxic species by a change in the ambient redox milieu. The details of this complex redox chemistry of NO may provide a mechanism for harnessing neuroprotective effects and avoiding neurotoxicity in the central nervous system.
MEF2C, a MADS/MEF2-family transcription factor expressed in a laminar distribution in cerebral cortex.
We have cloned cDNA encoding a human transcription factor that belongs to the MEF2 (myocyte-specific enhancer-binding factor 2) subfamily of the MADS (MCM1-agamous-deficiens-serum response factor) gene family. This factor, which we have named MEF2C, binds specifically to the MEF2 element and activates transcription via this element. Specific isoforms of this factor are found exclusively in brain and are robustly expressed by neurons in cerebral cortex. In situ hybridization indicates that the factor is expressed preferentially in certain neuronal layers of cortex and that expression declines during postnatal development. The unusual pattern of expression in brain suggests that this transcription factor may be important in the development of cortical architecture.
Human immunodeficiency virus-infected macrophages, gp120, and N-methyl-D-aspartate receptor-mediated neurotoxicity.
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Delayed administration of memantine prevents N-methyl-D-aspartate receptor-mediated neurotoxicity.
Increasing evidence supports the hypothesis that escalating levels of excitatory amino acids (EAAs) are responsible for neuronal cell death in a variety of acute neurological conditions including hypoxia/ischemia, trauma, seizures, and hypoglycemia. EAAs may also contribute to several chronic neurodegenerative diseases including Huntington's disease, parkinsonism, and acquired immunodeficiency syndrome dementia. A predominant form of neurotoxicity appears to be mediated by excessive activation of the N-methyl-D-aspartate subtype of glutamate receptor. This laboratory recently reported that memantine, an antiparkinsonian drug, is a potent N-methyl-D-aspartate antagonist capable of preventing the death of central neurons both in vitro and in vivo when given coincident to an EAA insult. In the present study, we found that 12 microM memantine prevented the death of neonatal rat retinal ganglion cells in primary culture when administered up to 4 hours after the initiation of N-methyl-D-aspartate receptor-mediated neurotoxicity.
Prospects for clinically tolerated NMDA antagonists: open-channel blockers and alternative redox states of nitric oxide.
Several acute and chronic neurological diseases might be mediated, at least in part, via stimulation of excitatory amino acid receptors, such as the N-methyl-D-aspartate (NMDA) receptor. Antagonists of excitatory amino acid receptors ameliorate neurotoxic damage in several animal models of these disorders. This review focuses on the potential for clinically tolerated NMDA receptor antagonists, with emphasis on agents that have been in clinical use for other conditions and that recently have been shown to inhibit NMDA receptor activity by a mechanism of open-channel block or redox modification.
hMEF2C gene encodes skeletal muscle- and brain-specific transcription factors.
The myocyte enhancer-binding factor 2 (MEF2) site is an essential element of many muscle-specific enhancers and promoters that binds nuclear proteins from muscle and brain. Recently, we have cloned a family of MEF2 transcription factors produced by two genes that, at the mRNA level, are broadly expressed and produce tissue-specific isoforms by posttranscriptional processes (Y.-T. Yu, R. E. Breitbart, L. B. Smoot, Y. Lee, V. Mahdavi, and B. Nadal-Ginard, Genes Dev. 6:1783-1798, 1992). Here, we report the isolation and functional characterization of cDNA clones encoding four MEF2 factors derived from a separate gene that we have named hMEF2C. In contrast to those of the previously reported genes, the transcripts of the hMEF2C gene are restricted to skeletal muscle and brain. One of the alternate exons is exclusively present in brain transcripts. The products of this gene have DNA-binding and trans-activating activities indistinguishable from those of the previously reported MEF2 factors. The hMEF2C gene is induced late during myogenic differentiation, and its expression is limited to a subset of cortical neurons. The potential targets for this transcription factor in a subset of neurons are not known at this time. The strict tissue-specific pattern of expression of hMEF2C in comparison with the more ubiquitous expression of other MEF2 genes suggests a different mode of regulation and a potentially important role of hMEF2C factors in myogenesis and neurogenesis.
Molecular mechanisms of trauma-induced neuronal degeneration.
Recent studies suggest that various neuron-specific responses, including activation of glutamate receptors, muscarinic receptors, and oxidative stress, contribute to the events leading to brain and spinal cord neuronal degeneration after trauma. These specific mechanisms and possible future treatments are highlighted in this review.
Blockade of NMDA receptor-mediated mobilization of intracellular Ca2+ prevents neurotoxicity.
NMDA receptor activation leads to elevated Ca2+ in cultured rat cortical and retinal ganglion cell neurons. If excessive, this Ca2+ response is associated with delayed neurotoxicity. We used dantrolene and ionomycin to test if the Ca2+ response to NMDA was due to mobilization of intracellular Ca2+ stores rather than merely to Ca2+ influx. In the presence of EGTA, ionomycin resulted in release and subsequent depletion of intracellular Ca2+ stores. Henceforth, despite normal extracellular Ca2+, NMDA elicited only about half of its former Ca2+ response. Similarly, when dantrolene was used to block Ca2+ release from intracellular stores, we observed > 50% smaller NMDA-evoked Ca2+ responses. These results quite surprisingly indicate that at least half the Ca2+ response to NMDA is due to release of intracellular Ca2+, a process triggered by influx of extracellular Ca2+. Dantrolene also protected neurons from NMDA receptor-mediated neurotoxicity. Release of intracellular Ca2+ may therefore be a necessary step in the cascade leading to neuronal damage induced by excessive NMDA receptor stimulation and may be amenable to pharmacological intervention.
L-homocysteic acid selectively activates N-methyl-D-aspartate receptors of rat retinal ganglion cells.
To explore the possible role of L-homocysteic acid (HCA) as a retinal transmitter, whole-cell recordings with patch electrodes were performed on isolated rat retinal ganglion cells (RGCs) in culture. HCA elicited an inward current at -60 mV. Similar to currents evoked at this potential in RGCs by N-methyl-D-aspartate (NMDA), HCA-activated currents were of relatively small amplitude (10-150 pA), and the noise level increased dramatically during the response. When HCA was co-applied with concentrations of NMDA that elicited a maximal response, the current was not increased over that of NMDA alone. HCA-evoked currents were almost completely blocked by the NMDA antagonists D-2-amino-5-phosphonovalerate (D-AP5), Mg2+, or 7-chlorokynurenate (7-Cl KYN). Unlike its effects on other preparations, even millimolar concentrations of HCA did not activate kainate-like currents. These observations suggest that HCA specifically activates the NMDA receptor-channel complex of rat RGCs.
Selectivity of Thy-1 monoclonal antibodies in enhancing neurite outgrowth.
Thy-1 monoclonal antibodies (MAbs) have previously been shown to promote neurite outgrowth from retinal ganglion cells and a variety of other neurons. We have studied the effect on neurite outgrowth of several Thy-1 MAbs with quantitatively similar binding properties and found that only certain Thy-1 MAbs promote neurite outgrowth. This finding suggests that the antibody effects depend on specific interactions with one or more active sites on the Thy-1 glycoprotein.
Models of neuronal injury in AIDS: another role for the NMDA receptor?
As many as two-thirds of patients with acquired immunodeficiency syndrome (AIDS) eventually suffer from neurological manifestations, including dysfunction of cognition, movement and sensation. How can human immunodeficiency virus type 1 (HIV-1) result in neuronal damage if neurons themselves are not infected by the virus? In this article Stuart Lipton reviews a series of experiments from several different laboratories that offer related hypotheses accounting for neurotoxicity in the brains of AIDS patients. There is growing support for the existence of HIV- or immune-related toxins that directly or indirectly lead to the injury or demise of neurons via a potentially complex web of interactions between macrophages (or microglia), astrocytes and neurons. However, a final common pathway for neuronal susceptibility appears to be operative, similar to that observed after stroke, trauma and epilepsy. This mechanism involves voltage-dependent Ca2+ channels and NMDA receptor-operated channels, and therefore offers hope for future pharmacological intervention.
Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex.
Nitric oxide (NO) is an important messenger both systemically and in the CNS. In digital Ca2+ imaging and patch-clamp experiments, clinically available nitroso compounds that generate NO are shown to inhibit responses mediated by the NMDA subtype of the glutamate receptor on rat cortical neurons in vitro. A mechanism of action for this effect was investigated by using the specific NO-generating agent S-nitrosocysteine. We propose that free sulfhydryl groups on the NMDA receptor-channel complex react to form one or more S-nitrosothiols in the presence of NO. If vicinal thiol groups react in this manner, they can form a disulfide bond(s), which is thought to constitute the redox modulatory site of the receptor, resulting in a relatively persistent blockade of NMDA responses. These reactions with NO can afford protection from NMDA receptor-mediated neurotoxicity. Our results demonstrate a new pathway for NO regulation of physiological function that is not via cGMP, but instead involves reactions with membrane-bound thiol groups on the NMDA receptor-channel complex.
Entubulization repair of severed cavernous nerves in the rat resulting in return of erectile function.
Erectile dysfunction is a significant complication of radical pelvic surgery in men. Using the rat as an experimental model, we investigated the feasibility of repairing surgically ablated cavernous nerves utilizing silastic tube nerve growth conduits filled with nerve growth enhancing media. Known fertile male Sprague-Dawley rats were randomly divided into four surgical groups consisting of nerve ablation, immediate nerve reconstruction utilizing the entubulization technique (two groups) and control. The nerve ablation group had five mm. sections of the cavernosal nerve excised bilaterally. The entubulization nerve graft group had five mm. sections of the cavernous nerve excised bilaterally, followed by immediate microsurgical reconstruction with a silastic nerve tube conduit filled with either Matrigel and heparin (MA) or Matrigel and heparin plus acidic fibroblast growth factor (MA/aFGF), interposed between the severed cavernous nerve stumps bilaterally. The control group underwent sham operations with the cavernous nerves being exposed only. Erectile function was evaluated at one, two, and four months postoperatively. Return of erectile function was defined as tumescence of the corporal bodies with application of direct electrical stimulation (four volts of five millisecond pulses at 20 Hertz) to the proximal cavernous nerves. The two and four month electrical stimulation studies resulted in tumescence from 50% and 58% of the entubulization nerve reconstructed nerves with MA/aFGF versus 29% and 30% for the MA only group and only 5% and 11% for the ablated group, respectively. We conclude that in this experimental model immediate nerve graft repair utilizing entubulization techniques with the addition of nerve growth enhancing media appears to be a successful method of salvaging erectile function when the cavernous nerves have been divided.
A slowly inactivating K+ current in retinal ganglion cells from postnatal rat.
The whole-cell configuration of the patch-clamp technique was used to study voltage-gated K+ conductances in retinal ganglion cells from postnatal rat. Retinal ganglion cells were fluorescently labeled in situ, dissociated from the retina, and maintained in culture. With physiological solutions in the bath and the pipette, depolarizing voltage steps from physiological holding potentials activated Na(+)-(INa), Ca2+ (ICa), and K(+)-currents studied previously in retinal ganglion cells. Here we report on a slowly decaying K+ current, not heretofore reported in rat. With 4-AP, TEA, and Co2+ in the bath, to block IA, IK, and IK(Ca), respectively, a slowly decaying outward current was activated from -80 mV by steps positive to -40 mV. This current was present in 92% of all ganglion cells tested (n = 83). It activated within 10 ms and inactivated with a voltage-independent time constant of about 70 ms at 35 degrees C. Inactivation was voltage-dependent, half-maximal at -55 mV, and almost complete at 0 mV. The current was blocked by internal Cs+ and TEA, or by external application of 1 mM Ba2+, but not by 3 mM extracellular Co2+. The biophysical and pharmacological properties of this current are distinctly different from those of slowly inactivating K+ currents studied in other rat neurons. It was very similar, however, to a slowly inactivating K+ current previously reported in ganglion cells of tiger salamander retina. This last finding indicates conservation of a defined K+ channel type in functionally related cells in both lower vertebrates and mammals.
Requirement for macrophages in neuronal injury induced by HIV envelope protein gp120.
HIV-1-related neuronal injury may involve a complex web of viral proteins and cytokines, but neurons themselves are not infected. The HIV envelope protein gp120 has been shown to engender an early increase in neuronal free calcium followed by delayed excitotoxic-like damage, which is prevented by N-methyl-D-aspartate (NMDA) antagonists. In the present study, we found that the injurious effects of gp120 on retinal ganglion cell neurons require the presence of macrophages in mixed neuronal glial cultures of postnatal retina. Within 24 hours of incubation, 20 pM gp120 injured nearly 40% of retinal ganglion cells in cultures containing macrophages and other glial cells, whereas no deleterious effects of gp120 were noted on retinal ganglion cells in cultures depleted of macrophages. Thus, the toxic effect of gp120 on neurons appears to be an indirect one, mediated by activation of macrophages and perhaps other glial cells.
Memantine prevents HIV coat protein-induced neuronal injury in vitro.
Studies with in vitro model systems suggest that at least part of the neurologic deficits of human immunodeficiency virus (HIV)-1-associated cognitive/motor complex may stem from neuronal injury mediated by the HIV-1 coat protein gp120. Concurrent activation of N-methyl-D-aspartate (NMDA) receptors is also necessary for gp120 to induce neuronal damage. We studied memantine, a drug that blocks NMDA receptor-operated ion channels, for possible protective effects from gp120-induced neuronal injury. In identified rat retinal ganglion cells in culture, we found that 2 microM memantine completely prevented the injury engendered by 20 pM gp120. These data suggest that memantine has therapeutic potential as an NMDA antagonist capable of ameliorating neuronal damage associated with gp120.