Is tissue plasminogen activator a threat to neurons?

The clot-busting drug tissue plasminogen activator (tPA) is currently the only FDA-approved therapy for acute stroke. However, increasing evidence suggests that tPA can also contribute to excitotoxic neuronal damage in animal models of stroke.

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.

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.

Nicotinic antagonists enhance process outgrowth by rat retinal ganglion cells in culture.

Functional nicotinic cholinergic receptors are found on mammalian retinal ganglion cell neurons in culture. The neurotransmitter acetylcholine (ACh) can be detected in the medium of many of these retinal cultures, after release presumably from the choline acetyltransferase-positive amacrine cells. The postsynaptic effect of endogenous or applied ACh on the ganglion cells can be blocked with specific nicotinic antagonists. Here it is shown that within 24 hours of producing such a pharmacologic blockade, the retinal ganglion cells begin to sprout or regenerate neuronal processes. Thus, the growth-enhancing effect of nicotinic antagonists may be due to the removal of inhibition to growth by tonic levels of ACh present in the culture medium. Since there is a spontaneous leak of ACh in the intact retina, the effects of nicotinic cholinergic drugs on process outgrowth in culture may reflect a normal control mechanism for growth or regeneration of retinal ganglion cell processes that is exerted by ACh in vivo.

HIV-1 coat protein neurotoxicity prevented by calcium channel antagonists.

Coat protein gp120 from the human immunodeficiency virus type-1 (HIV-1) increased intracellular free calcium and injured rodent retinal ganglion cells and hippocampal neurons in culture. Highly purified recombinant gp120 envelope protein produced these effects in a dose-dependent fashion at picomolar concentrations. Immunoprecipitation with antibody to gp120, but not with control immunoglobulin-containing serum, depleted solutions of the viral envelope protein and also prevented both the rise in intracellular calcium and neuronal toxicity. The gp120-induced increase in intracellular calcium was abrogated by transiently lowering extracellular calcium or by adding the dihydropyridine calcium channel antagonist nimodipine (100 nM). Calcium channel antagonists also prevented gp120-induced neuronal injury. In addition, intracellular stores appeared to contribute substantially to the increase in calcium elicited by gp120. Since increases in intracellular calcium have been associated with neurotoxicity, it is possible that an injurious effect of gp120 on neurons might be related to this mechanism and that treatment with calcium channel antagonists may prove useful in mitigating HIV-1-related neuronal injury.

Monoclonal antibody to Thy-1 enhances regeneration of processes by rat retinal ganglion cells in culture.

Ganglion cells were dissociated from postnatal rat retinas, identified by specific fluorescent labels, and maintained in culture on a variety of substrates. Regeneration of processes by retinal ganglion cells was enhanced when the cells were plated on glass coated with a monoclonal antibody against the Thy-1 determinant. Plain glass and glass coated with polylysine, collagen, fibronectin, or other monoclonal antibodies supported the growth of neural processes, but were less effective than antibody to Thy-1.

VIP-mediated increase in cAMP prevents tetrodotoxin-induced retinal ganglion cell death in vitro.

Afferent influences on natural cell death were modeled in retinal cultures derived from neonatal rats. Tetrodotoxin (TTX) blockade of electrical activity produced a significant reduction in surviving retinal ganglion cell (RGC) neurons during a critical period of development, similar in magnitude to the reduction observed during natural cell death in the intact retina at a similar developmental stage. The addition of vasoactive intestinal peptide (VIP) protected the RGCs from the lethal action of TTX. This effect was specific, since the related peptides PHI-27 and secretin produced no significant increase in RGC survival. Radioimmunoassay of cyclic nucleotides showed that TTX decreased culture levels of cAMP and that this trend was reversed by VIP. Decreases in RGC survival associated with TTX electrical blockade were prevented by 8-bromo:cAMP or forskolin. Furthermore, VIP10-28, the C-terminal fragment that inhibits VIP stimulation of adenylate cyclase, reduced the number of surviving RGCs. Thus, our results suggest that VIP, acting by increasing cAMP, has a neurotrophic effect on electrically blocked RGCs and may be an endogenous factor modulating normal cell death in the retina.

Identification and mechanism of action of two histidine residues underlying high-affinity Zn2+ inhibition of the NMDA receptor.

Zinc (Zn2+) inhibition of N-methyl-D-aspartate receptor (NMDAR) activity involves both voltage-independent and voltage-dependent components. Recombinant NR1/NR2A and NR1/NR2B receptors exhibit similar voltage-dependent block, but voltage-independent Zn2+ inhibition occurs with much higher affinity for NR1/NR2A than NR1/NR2B receptors (nanomolar versus micromolar IC50, respectively). Here, we show that two neighboring histidine residues on NR2A represent the critical determinant (termed the "short spacer") for high-affinity, voltage-independent Zn2+ inhibition using the Xenopus oocyte expression system and site-directed mutagenesis. Mutation of either one of these two histidine residues (H42 and H44) in the extracellular N-terminal domain of NR2A shifted the IC50 for high-affinity Zn2+ inhibition approximately 200-fold without affecting the EC50 of the coagonists NMDA and glycine. We suggest that the mechanism of high-affinity Zn2+ inhibition on the NMDAR involves enhancement of proton inhibition.

Selective modulation of NMDA responses by reduction and oxidation.

Electrophysiological responses to the glutamate analog N-methyl-D-aspartate (NMDA) measured in three different central neuronal preparations are subject to a novel modulatory mechanism: they are substantially potentiated after exposure to the disulfide reducing agent dithiothreitol, while oxidation with 5-5-dithiobis-2-nitrobenzoic acid decreases the magnitude of the response. Modification of the NMDA response by either oxidation or reduction does not appear to affect the pharmacological properties of the receptor-channel complex. Since we observe that the redox state of the native receptor-channel complex varies widely among neurons, an in vivo mechanism that can strongly regulate NMDA-activated functions by either reduction or oxidation may exist. In addition, these results suggest that it may be possible to design specific redox agents for characterizing the NMDA receptor-channel complex.

Synergistic effects of HIV coat protein and NMDA receptor-mediated neurotoxicity.

Exposure of rat retinal cultures to HIV-1 coat protein gp120 for several minutes increases [Ca2+]i in approximately half of the ganglion cells; this effect is associated with delayed-onset neuronal injury, similar to that previously reported in NMDA receptor-mediated neurotoxicity. Here we show that NMDA antagonists can prevent both the rise in [Ca2+]i and subsequent neuronal damage engendered by 20 pM gp120. However, whole-cell patch-clamp recordings demonstrate that gp120 does not directly evoke an NMDA-like response or enhance glutamate/NMDA-activated currents. Moreover, complete protection from gp120-induced [Ca2+]i increases and neurotoxicity is afforded by incubation with glutamate-pyruvate transaminase, which breaks down endogenous glutamate as verified by HPLC. Since, under standard conditions in these cultures, neither glutamate nor a low picomolar concentration of gp120 is deleterious on its own, our results suggest that their neurotoxicity is synergistic.

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.

Identification of two cysteine residues that are required for redox modulation of the NMDA subtype of glutamate receptor.

Modulation of NMDA-mediated responses by oxidizing and reducing reagents has been described in a variety of neuronal preparations. Here, we report that NMDA-gated currents of oocytes expressing heteromeric NMDA receptors are also modulated by sulfhydryl redox reagents. Each cysteine residue in the NMDAR1 (NR1) subunit and each conserved NMDAR2 (NR2) cysteine residue in a prototypical subunit (NR2B) was tested for its role in redox modulation. We have identified 2 cysteines in the NR1 subunit that are required for redox modulation of NMDA-gated currents in oocytes expressing NR1-NR2B, NR1-NR2C, or NR1-NR2D receptors. Mutation of these same 2 cysteines also eliminated potentiation by spermine and shifted the IC50 for H+ inhibition and the EC50 for NMDA. Redox modulation of heteromeric NR1-NR2A receptors appeared to be different from that of the other heteromeric receptors, indicating the presence of one or more unique redox modulatory sites on NR1-NR2A receptors.

Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function.

During ischemic brain injury, glutamate accumulation leads to overstimulation of postsynaptic glutamate receptors with intracellular Ca2+ overload and neuronal cell death. Here we show that glutamate can induce either early necrosis or delayed apoptosis in cultures of cerebellar granule cells. During and shortly after exposure to glutamate, a subpopulation of neurons died by necrosis. In these cells, mitochondrial membrane potential collapsed, nuclei swelled, and intracellular debris were scattered in the incubation medium. Neurons surviving the early necrotic phase recovered mitochondrial potential and energy levels. Later, they underwent apoptosis, as shown by the formation of apoptotic nuclei and by chromatin degradation into high and low molecular weight fragments. These results suggest that mitochondrial function is a critical factor that determines the mode of neuronal death in excitotoxicity.

Attenuation of NMDA receptor activity and neurotoxicity by nitroxyl anion, NO-.

Recent evidence indicates that the NO-related species, nitroxyl anion (NO), is produced in physiological systems by several redox metal-containing proteins, including hemoglobin, nitric oxide synthase (NOS), superoxide dismutase, and S-nitrosothiols (SNOs), which have recently been identified in brain. However, the chemical biology of NO- remains largely unknown. Here, we show that NO- -unlike NO*, but reminiscent of NO+ transfer (or S-nitrosylation)- -reacts mainly with Cys-399 in the NR2A subunit of the N-methyl-D-aspartate (NMDA) receptor to curtail excessive Ca2+ influx and thus provide neuroprotection from excitotoxic insults. This effect of NO- closely resembles that of NOS, which also downregulates NMDA receptor activity under similar conditions in culture.

S-Nitrosylation and uncompetitive/fast off-rate (UFO) drug therapy in neurodegenerative disorders of protein misfolding.

Although activation of glutamate receptors is essential for normal brain function, excessive activity leads to a form of neurotoxicity known as excitotoxicity. Key mediators of excitotoxic damage include overactivation of N-methyl-D-aspartate (NMDA) receptors, resulting in excessive Ca(2+) influx with production of free radicals and other injurious pathways. Overproduction of free radical nitric oxide (NO) contributes to acute and chronic neurodegenerative disorders. NO can react with cysteine thiol groups to form S-nitrosothiols and thus change protein function. S-nitrosylation can result in neuroprotective or neurodestructive consequences depending on the protein involved. Many neurodegenerative diseases manifest conformational changes in proteins that result in misfolding and aggregation. Our recent studies have linked nitrosative stress to protein misfolding and neuronal cell death. Molecular chaperones - such as protein-disulfide isomerase, glucose-regulated protein 78, and heat-shock proteins - can provide neuroprotection by facilitating proper protein folding. Here, we review the effect of S-nitrosylation on protein function under excitotoxic conditions, and present evidence that NO contributes to degenerative conditions by S-nitrosylating-specific chaperones that would otherwise prevent accumulation of misfolded proteins and neuronal cell death. In contrast, we also review therapeutics that can abrogate excitotoxic damage by preventing excessive NMDA receptor activity, in part via S-nitrosylation of this receptor to curtail excessive activity.

HIV-1 coreceptors CCR5 and CXCR4 both mediate neuronal cell death but CCR5 paradoxically can also contribute to protection.

The chemokine receptors CCR5 and CXCR4 serve, in addition to CD4, as coreceptors for human immunodeficiency virus-1 (HIV-1), and infection with HIV-1 can cause dementia. In brain-derived cells, HIV-1 envelope glycoprotein gp120 initiates a signaling cascade that involves p38 mitogen-activated protein kinase and leads to neuronal cell death. Using mixed neuronal/glial cultures from rats and mice genetically deficient in one or both HIV coreceptors, we show here that CCR5, CXCR4 or both can mediate HIV/gp120 neurotoxicity depending on the viral strain. Paradoxically, we also found evidence for a CCR5-mediated neuroprotective pathway. We identify protein kinase Akt/PKB as an essential component of this pathway, which can be triggered by the CCR5 agonists macrophage inflammatory protein-1beta and regulated-and-normal-T-cell-expressed-and-secreted. Moreover, these CCR5 ligands prevent neuronal cell death induced by stromal cell-derived factor-1, a CXCR4 agonist. Both neurons and glia coexpress CXCR4 and CCR5. Ca2+ imaging experiments demonstrate that engagement of CCR5 prevents CXCR4-triggered increases in intracellular free Ca2+. This finding suggests that CCR5 ligands can protect neurons at least, in part, by modulating CXCR4-mediated toxicity through heterologous desensitization.

Mitochondrial fission is an upstream and required event for bax foci formation in response to nitric oxide in cortical neurons.

Mitochondrial dysfunction is an underpinning event in many neurodegenerative disorders. Less clear, however, is how mitochondria become injured during neuronal demise. Nitric oxide (NO) evokes rapid mitochondrial fission in cortical neurons. Interestingly, proapoptotic Bax relocates from the cytoplasm into large foci on mitochondrial scission sites in response to nitrosative stress. Antiapoptotic Bcl-xL does not prevent mitochondrial fission despite its ability to block Bax puncta formation on mitochondria and to mitigate neuronal cell death. Mitofusin 1 (Mfn1) or dominant-negative dynamin-related protein 1(K38A) (Drp1(k38A)) inhibits mitochondrial fission and Bax accumulation on mitochondria induced by exposure to an NO donor. Although NO is known to cause a bioenergetic crisis, lowering ATP by glycolytic or mitochondrial inhibitors neither induces mitochondrial fission nor Bax foci formation on mitochondria. Taken together, these data indicate that the mitochondrial fission machinery acts upstream of the Bcl-2 family of proteins in neurons challenged with nitrosative stress.

Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation.

Several ion channels are thought to be directly modulated by nitric oxide (NO), but the molecular basis of this regulation is unclear. Here we show that the NMDA receptor (NMDAR)-associated ion channel was modulated not only by exogenous NO but also by endogenous NO. Site-directed mutagenesis identified a critical cysteine residue (Cys 399) on the NR2A subunit whose S-nitrosylation (NO+ transfer) under physiological conditions underlies this modulation. In cell systems expressing NMDARs with mutant NR2A subunits in which this single cysteine was replaced by an alanine, the effect of endogenous NO was lost. Thus endogenous S-nitrosylation can regulate ion channel activity.

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.

HIV-1 infection and AIDS: consequences for the central nervous system.

Infection with the human immunodeficiency virus-1 (HIV-1) can induce severe and debilitating neurological problems that include behavioral abnormalities, motor dysfunction and frank dementia. After infiltrating peripheral immune competent cells, in particular macrophages, HIV-1 provokes a neuropathological response involving all cell types in the brain. HIV-1 also incites activation of chemokine receptors, inflammatory mediators, extracellular matrix-degrading enzymes and glutamate receptor-mediated excitotoxicity, all of which can trigger numerous downstream signaling pathways and disrupt neuronal and glial function. This review will discuss recently uncovered pathologic neuroimmune and degenerative mechanisms contributing to neuronal damage induced by HIV-1 and potential approaches for development of future therapeutic intervention.