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Over-expression of mutant copper, zinc superoxide dismutase (SOD) in mice induces ALS and has become the most widely used model of neurodegeneration. However, no pharmaceutical agent in 20 years has extended lifespan by more than a few weeks. The Copper-Chaperone-for-SOD (CCS) protein completes the maturation of SOD by inserting copper, but paradoxically human CCS causes mice co-expressing mutant SOD to die within two weeks of birth. Hypothesizing that co-expression of CCS created copper deficiency in spinal cord, we treated these pups with the PET-imaging agent CuATSM, which is known to deliver copper into the CNS within minutes. CuATSM prevented the early mortality of CCSxSOD mice, while markedly increasing Cu, Zn SOD protein in their ventral spinal cord. Remarkably, continued treatment with CuATSM extended the survival of these mice by an average of 18 months. When CuATSM treatment was stopped, these mice developed ALS-related symptoms and died within 3 months. Restoring CuATSM treatment could rescue these mice after they became symptomatic, providing a means to start and stop disease progression. All ALS patients also express human CCS, raising the hope that familial SOD ALS patients could respond to CuATSM treatment similarly to the CCSxSOD mice., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Peroxynitrite production and tyrosine nitration are present in several pathological conditions, including neurodegeneration, stroke, aging, and cancer. Nitration of the pro-survival chaperone heat shock protein 90 (Hsp90) in position 33 and 56 induces motor neuron death through a toxic gain-of-function. Here we show that nitrated Hsp90 regulates mitochondrial metabolism independently of the induction of cell death. In PC12 cells, a small fraction of nitrated Hsp90 was located on the mitochondrial outer membrane and down-regulated mitochondrial membrane potential, oxygen consumption, and ATP production. Neither endogenous Hsp90 present in the homogenate nor unmodified and fully active recombinant Hsp90 was able to compete with the nitrated protein for the binding to mitochondria. Moreover, endogenous or recombinant Hsp90 did not prevent the decrease in mitochondrial activity but supported nitrated Hsp90 mitochondrial gain-of-function. Nitrotyrosine in position 33, but not in any of the other four tyrosine residues prone to nitration in Hsp90, was sufficient to down-regulate mitochondrial activity. Thus, in addition to induction of cell death, nitrated Hsp90 can also regulate mitochondrial metabolism, suggesting that depending on the cell type, distinct Hsp90 nitration states regulate different aspects of cellular metabolism. This regulation of mitochondrial homeostasis by nitrated Hsp90 could be of particular relevance in cancer cells., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Nitrotyrosine is used as a marker for the production of peroxynitrite and other reactive nitrogen species. For over 20 years the presence of nitrotyrosine was associated with cell death in multiple pathologies. Filling the gap between correlation and causality has proven to be a difficult task. Here, we discuss the evidence supporting tyrosine nitration as a specific posttranslational modification participating in the induction of cell death signaling pathways.

Oxidative stress is a widely recognized cause of cell death associated with neurodegeneration, inflammation, and aging. Tyrosine nitration in these conditions has been reported extensively, but whether tyrosine nitration is a marker or plays a role in the cell-death processes was unknown. Here, we show that nitration of a single tyrosine residue on a small proportion of 90-kDa heat-shock protein (Hsp90), is sufficient to induce motor neuron death by the P2X7 receptor-dependent activation of the Fas pathway. Nitrotyrosine at position 33 or 56 stimulates a toxic gain of function that turns Hsp90 into a toxic protein. Using an antibody that recognizes the nitrated Hsp90, we found immunoreactivity in motor neurons of patients with amyotrophic lateral sclerosis, in an animal model of amyotrophic lateral sclerosis, and after experimental spinal cord injury. Our findings reveal that cell death can be triggered by nitration of a single protein and highlight nitrated Hsp90 as a potential target for the development of effective therapies for a large number of pathologies.

Tyrosine nitration is a biomarker for the production of peroxynitrite and other reactive nitrogen species. Nitrotyrosine immunoreactivity is present in many pathological conditions including several cardiac diseases. Because the events observed during heart failure may recapitulate some aspects of development, we tested whether nitrotyrosine is present during normal development of the rat embryo heart and its potential relationship in cardiac remodeling through apoptosis. Nitric oxide production is highly dynamic during development, but whether peroxynitrite and nitrotyrosine are formed during normal embryonic development has received little attention. Rat embryo hearts exhibited strong nitrotyrosine immunoreactivity in endocardial and myocardial cells of the atria and ventricles from E12 to E18. After E18, nitrotyrosine staining faded and disappeared entirely by birth. Tyrosine nitration in the myocardial tissue coincided with elevated protein expression of nitric oxide synthases (eNOS and iNOS). The immunoreactivity for these NOS isoforms remained elevated even after nitrotyrosine had disappeared. Tyrosine nitration did not correlate with cell death or proliferation of cardiac cells. Analysis of tryptic peptides by MALDI-TOF showed that nitration occurs in actin, myosin, and the mitochondrial ATP synthase α chain. These results suggest that reactive nitrogen species are not restricted to pathological conditions but may play a role during normal embryonic development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Oxidative damage is a common and early feature of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and other neurodegenerative disorders. Dr. Mark Smith and his colleagues have built the case for oxidative stress being a primary progenitor rather than a secondary end-stage epiphenomenon of neurodegeneration. They proposed that reactive oxygen species contribute to the "age-related cascade of neurodegeneration," whereby accumulative oxidative damage with age promotes other characteristic pathological changes in afflicted brain regions, including protein aggregation, metabolic deficiencies, and inflammation. Nitric oxide (NO) likely plays a critical role in this age-related cascade. NO is a major signaling molecule produced in the central nervous system to modulate neurological activity through stimulating cyclic GMP synthesis. However, the same physiological concentrations of NO, relevant in cellular signaling, may also initiate and amplify oxidative damage by diffusion-limited reactions with superoxide (O(2)(•-)) to produce peroxynitrite (ONOO(-)). This is perhaps best illustrated in ALS where physiological levels of NO promote survival of motor neurons, but the same concentrations can stimulate motor neuron apoptosis and glial cell activation under pathological conditions. While these changes represent a complex mechanism involving multiple cell types in the pathogenesis of ALS, they also reveal general processes underlying neurodegeneration.

When replete with zinc and copper, amyotrophic lateral sclerosis (ALS)-associated mutant SOD proteins can protect motor neurons in culture from trophic factor deprivation as efficiently as wild-type SOD. However, the removal of zinc from either mutant or wild-type SOD results in apoptosis of motor neurons through a copper- and peroxynitrite-dependent mechanism. It has also been shown that motor neurons isolated from transgenic mice expressing mutant SODs survive well in culture but undergo apoptosis when exposed to nitric oxide via a Fas-dependent mechanism. We combined these two parallel approaches for understanding SOD toxicity in ALS and found that zinc-deficient SOD-induced motor neuron death required Fas activation, whereas the nitric oxide-dependent death of G93A SOD-expressing motor neurons required copper and involved peroxynitrite formation. Surprisingly, motor neuron death doubled when Cu,Zn-SOD protein was either delivered intracellularly to G93A SOD-expressing motor neurons or co-delivered with zinc-deficient SOD to nontransgenic motor neurons. These results could be rationalized by biophysical data showing that heterodimer formation of Cu,Zn-SOD with zinc-deficient SOD prevented the monomerization and subsequent aggregation of zinc-deficient SOD under thiol-reducing conditions. ALS mutant SOD was also stabilized by mutating cysteine 111 to serine, which greatly increased the toxicity of zinc-deficient SOD. Thus, stabilization of ALS mutant SOD by two different approaches augmented its toxicity to motor neurons. Taken together, these results are consistent with copper-containing zinc-deficient SOD being the elusive "partially unfolded intermediate" responsible for the toxic gain of function conferred by ALS mutant SOD.

Depending on its concentration, nitric oxide (NO) has beneficial or toxic effects. In pathological conditions, NO reacts with superoxide to form peroxynitrite, which nitrates proteins forming nitrotyrosine residues (3NY), leading to loss of protein function, perturbation of signal transduction, and cell death. 3NY immunoreactivity is present in many CNS diseases, particularly multiple sclerosis. Here, using the high flux NO donor, spermine-NONOate, we report that oligodendrocytes are resistant to NO, while motor neurons are NO sensitive. Motor neuron sensitivity correlates with the NO-dependent formation of 3NY, which is significantly more pronounced in motor neurons when compared with oligodendrocytes, suggesting peroxynitrite as the toxic molecule. The heme-metabolizing enzyme, heme-oxygenase-1 (HO1), is necessary for oligodendrocyte NO resistance, as demonstrated by loss of resistance after HO1 inhibition. Resistance is reinstated by peroxynitrite scavenging with uric acid further implicating peroxynitrite as responsible for NO sensitivity. Most importantly, differential sensitivity to NO is also present in cultures of primary oligodendrocytes and motor neurons. Finally, motor neurons cocultured with oligodendrocytes, or oligodendrocyte-conditioned media, become resistant to NO toxicity. Preliminary studies suggest oligodendrocytes release a soluble factor that protects motor neurons. Our findings challenge the current paradigm that oligodendrocytes are the exclusive target of multiple sclerosis pathology.

Vascular endothelial growth factor (VEGF) plays a neuroprotective role in mice harboring mutations of copper-zinc superoxide dismutase 1 (SOD1) in familial amyotrophic lateral sclerosis (ALS). Conversely, the loss of VEGF expression through genetic depletion can give rise to a phenotype resembling ALS independent of SOD1 mutations. Here, we observe a profound downregulation of VEGF mRNA expression in spinal cords of G93A SOD1 mice that occurred early in the course of the disease. Using an in vitro culture model of glial cells expressing mutant SOD1, we demonstrate destabilization and downregulation of VEGF RNA with concomitant loss of protein expression that correlates with level of transgene expression. Using a luciferase reporter assay, we show that this molecular effect is mediated through a portion of the VEGF 3'-untranslated region (UTR) that harbors a class II adenylate/uridylate-rich element. Other mutant forms of SOD1 produced a similar negative effect on luciferase RNA and protein expression. Mobility shift assay with a VEGF 3'-UTR probe reveals an aberrantly migrating complex that contains mutant SOD1. We further show that the RNA stabilizing protein, HuR (human antigen R), is translocated from nucleus to cytoplasm in mutant SOD1 cells in vitro and mouse motor neurons in vivo. In summary, our data suggest that mutant SOD1 gains a novel function, possibly by altering the ribonucleoprotein complex with the VEGF 3'-UTR. We postulate that the resultant dysregulation of VEGF posttranscriptional processing critically reduces the level of this neuroprotective growth factor and accelerates the neurodegenerative process in ALS.

Although peroxynitrite stimulates apoptosis in many cell types, whether peroxynitrite acts directly as an oxidant or the induction of apoptosis is because of the radicals derived from peroxynitrite decomposition remains unknown. Before undergoing apoptosis because of trophic factor deprivation, primary motor neuron cultures become immunoreactive for nitrotyrosine. We show here using tyrosine-containing peptides that free radical processes mediated by peroxynitrite decomposition products were required for triggering apoptosis in primary motor neurons and in PC12 cells cultures. The same concentrations of tyrosine-containing peptides required to prevent the nitration and apoptosis of motor neurons induced by trophic factor deprivation and of PC12 cells induced by peroxynitrite also prevented peroxynitrite-mediated nitration of motor neurons, brain homogenates, and PC12 cells. The heat shock protein 90 chaperone was nitrated in both trophic factor-deprived motor neurons and PC12 cells incubated with peroxynitrite. Tyrosine-containing peptides did not affect the induction of PC12 cell death by hydrogen peroxide. Tyrosine-containing peptides should protect by scavenging peroxynitrite-derived radicals and not by direct reactions with peroxynitrite as they neither increase the rate of peroxynitrite decomposition nor decrease the bimolecular peroxynitrite-mediated oxidation of thiols. These results reveal an important role for free radical-mediated nitration of tyrosine residues, in apoptosis induced by endogenously produced and exogenously added peroxynitrite; moreover, tyrosine-containing peptides may offer a novel strategy to neutralize the toxic effects of peroxynitrite.

The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.

When deprived of trophic factors, the majority of cultured motor neurons undergo nitric oxide-dependent apoptosis. However, for reasons that have remained unclear, 30-50% of the motor neurons survive for several days without trophic factors. Here we hypothesize that the resistance of this motor neuron subpopulation to trophic factor deprivation can be attributed to diminished nitric oxide production resulting from the activity of the arginine-degrading enzyme arginase. When incubated with nor-N(G)-hydroxy-nor-L-arginine (NOHA), the normally resistant trophic factor-deprived motor neurons showed a drop in survival rates, whereas trophic factor-treated neurons did not. NOHA-induced motor neuron death was inhibited by blocking nitric oxide synthesis and the scavenging of superoxide and peroxynitrite, suggesting that peroxynitrite mediates NOHA toxicity. When we transfected arginase 1 into motor neurons to see whether it alone could abrogate trophic factor deprivation-induced death, we found that its forced expression did indeed do so. The protection afforded by arginase 1 expression is reversed when cells are incubated with NOHA or with low concentrations of nitric oxide. These results reveal that arginase acts as a central regulator of trophic factor-deprived motor neuron survival by suppressing nitric oxide production and the consequent peroxynitrite toxicity. They also suggest that the resistance of motor neuron subpopulations to trophic factor deprivation may result from increased arginase activity.

Neuregulins play a major role in the formation and stabilization of neuromuscular junctions, and are produced by both motor neurons and muscle. Although the effects and mechanism of neuregulins on skeletal muscle (e.g. regulation of acetylcholine receptor expression) have been studied extensively, the effects of neuregulins on motor neurons remain unknown. We report that neuregulin-1beta (NRGbeta1) inhibited apoptosis of rat motor neurons for up to 7 days in culture by a phosphatidylinositol 3 kinase-dependent pathway and synergistically enhanced motor neuron survival promoted by glial-derived neurotrophic factor (GDNF). However, binding of neurotrophins, including brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), to the p75 neurotrophin receptor (p75NTR) abolished the neuregulin anti-apoptotic effect on motor neurons. Inhibitors of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase prevented motor neuron death caused by co-incubation of NRGbeta1 and BDNF or NGF, as well as by trophic factor deprivation. Motor neuron apoptosis resulting from both trophic factor deprivation and exposure to NRGbeta1 plus neurotrophins required the induction of neuronal nitric oxide synthase and peroxynitrite formation. Because motor neurons express both p75NTR and neuregulin erbB receptors during the period of embryonic programmed cell death, motor neuron survival may be the result of complex interactions between trophic and death factors, which may be the same molecules acting in different combinations.

Fibroblast growth factor-1 (FGF1 or acidic FGF) is highly expressed in motor neurons. FGF-1 is released from cells by oxidative stress, which might occur from SOD-1 aberrant function in amyotrophic lateral sclerosis (ALS). Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that FGF-1 could activate spinal cord astrocytes in a manner that decreased motor neuron survival in co-cultures. FGF-1 induced accumulation of the FGF receptor 1 (FGFR1) in astrocyte nuclei and potently stimulated nerve growth factor (NGF) expression and secretion. The FGFR1 tyrosine kinase inhibitor PD166866 prevented these effects. Previously, we have shown that NGF secretion by reactive astrocytes induces motor neuron apoptosis through a p75(NTR)-dependent mechanism. Embryonic motor neurons co-cultured on the top of astrocytes exhibiting activated FGFR1 underwent apoptosis, which was prevented by PD166866 or by adding either anti-NGF or anti-p75(NTR) neutralizing antibodies. In the degenerating spinal cord of mice carrying the ALS mutation G93A of Cu, Zn superoxide dismutase, FGF-1 was no longer localized only in the cytosol of motor neurons, while FGFR1 accumulated in the nuclei of reactive astrocytes. These results suggest that FGF-1 released by oxidative stress from motor neurons might have a role in activating astrocytes, which could in turn initiate motor neuron apoptosis in ALS through a p75(NTR)-dependent mechanism.

A strong glial reaction typically surrounds the affected upper and lower motor neurons and degenerating descending tracts of ALS patients. Reactive astrocytes in ALS contain protein inclusions, express inflammatory makers such as the inducible forms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2), display nitrotyrosine immunoreactivity and downregulate the glutamate transporter EAAT2. In this review, we discuss the evidence sustaining an active role for astrocytes in the induction and propagation of motor neuron loss in ALS. Available evidence supports the view that glial activation could be initiated by proinflammatory mediators secreted by motor neurons in response to injury, axotomy or muscular pathology. In turn, reactive astrocytes produce nitric oxide and peroxynitrite, which cause mitochondrial damage in cultured neurons and trigger apoptosis in motor neurons. Astrocytes may also contribute to the excitotoxic damage of motor neurons by decreasing glutamate transport or actively releasing the excitotoxic amino acid. In addition, reactive astrocytes secrete pro-apoptotic mediators, such as nerve growth factor (NGF) or Fas-ligand, a mechanism that may serve to eliminate vulnerable motor neurons. The comprehensive understanding of the interactions between motor neurons and glia in ALS may lead to a more accurate theory of the pathogenesis of the disease.

Background: Overproduction of nitric oxide (NO) has been recognized as a major mechanism of neurotoxicity. NO reacts with superoxide to generate peroxynitrite, a strong oxidizing and nitrating species. Peroxynitrite is formed in glial cells and degenerating neurons in neuropathological conditions, including amyotrophic lateral sclerosis (ALS). In ALS, motor neurons re-express the p75 neurotrophin receptor (p75NTR) and might become vulnerable to NGF. In the present study, we investigated whether peroxynitrite stimulated nerve growth factor (NGF) expression in spinal cord astrocytes., Materials and Methods: Astrocyte monolayers were exposed to peroxynitrite and nitrotyrosine formation was determined by immunofluorescence. mRNA levels for NGF, brain derived neutrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2) were measured by semi-quantitative RT-PCR and NGF release was determined by ELISA., Results and Discussion: A single exposure to peroxynitrite specifically induced NGF expression and secretion in astrocytes coincident with reactive morphological changes and increased nitrotyrosine immunoreactivity. These results suggest that NGF expression in reactive astrocytes is under the control of oxidative stress.

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75(NTR)) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75(NTR) was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75(NTR) blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.

Death pathways restricted to specific neuronal classes could potentially allow for precise control of developmental neuronal death and also underlie the selectivity of neuronal loss in neurodegenerative disease. We show that Fas-triggered death of normal embryonic motoneurons requires transcriptional upregulation of neuronal NOS and involves Daxx, ASK1, and p38 together with the classical FADD/caspase-8 cascade. No evidence for involvement of this pathway was found in cells other than motoneurons. Motoneurons from transgenic mice overexpressing ALS-linked SOD1 mutants (G37R, G85R, or G93A) displayed increased susceptibility to activation of this pathway: they were more sensitive to Fas- or NO-triggered cell death but not to trophic deprivation or excitotoxic stimulation. Thus, triggering of a motoneuron-restricted cell death pathway by neighboring cells might contribute to motoneuron loss in ALS.

Deprivation of trophic factors induces expression of neuronal nitric oxide synthase (NOS) and nitric oxide production in cultured motor neurons, leading to apoptosis. Motor neuron apoptosis requires the simultaneous production of nitric oxide and superoxide and is associated with increased nitrotyrosine immunoreactivity. Nitric oxide also stimulates cyclic guanosine 5' monophosphate (cGMP) synthesis, which enhances the survival of motor neurons treated with brain derived trophic factor (BDNF). Here we report that cGMP analogs blocked neuronal NOS induction, nitrotyrosine accumulation, and prevented apoptosis for up to 3 day of motor neurons deprived of trophic factors. Low concentrations of exogenous nitric oxide (<100 nM), which are not toxic for BDNF-treated cultures, reversed the protective effect of cGMP. These results suggest that elevation of cGMP could decrease nitric oxide production, and thereby preventing motor neuron apoptosis.

Free radicals and oxidants may have contrasting effects on cells. Nitric oxide is a cellular messenger that acts by several mechanisms, including activation of soluble guanylate cyclase, nitrosylation of thiols, and formation of peroxynitrite. The action of nitric oxide depends on oxidative conditions in the cell. In motor neurons, nitric oxide enhances brain-derived neurotrophic factor effects by stimulation of cGMP production. In the absence of trophic factors, nitric oxide induces motor neuron apoptosis by reacting with superoxide to form peroxynitrite. The mechanism of induction of motor neuron apoptosis by the formation of peroxynitrite is more complicated than simply the formation of a strong oxidant and it also involves decreased formation of cGMP.

Oxidative stress mediated by nitric oxide (NO) and its toxic metabolite peroxynitrite has previously been associated with motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Degenerating spinal motor neurons in familial and sporadic ALS are typically surrounded by reactive astrocytes expressing the inducible form of NO synthase (iNOS), suggesting that astroglia may have a pathogenic role in ALS. We report here that a brief exposure of spinal cord astrocyte monolayers to peroxynitrite (0.25-1 mM) provoked long-lasting reactive morphological changes characterized by process-bearing cells displaying intense glial fibrillary acidic protein and iNOS immunoreactivity. Furthermore, peroxynitrite caused astrocytes to promote apoptosis of embryonic motor neurons subsequently plated on the monolayers. Neuronal death occurred within 24 hr after plating, as evidenced by the presence of degenerating motor neurons positively stained for activated caspase-3 and nitrotyrosine. Motor neuron death was largely prevented by NOS inhibitors and peroxynitrite scavengers but not by trophic factors that otherwise will support motor neuron survival in the absence of astrocytes. The bacterial lipopolysaccharide, a well-known inflammatory stimulus that induces iNOS expression in astrocytes, provoked the same effects on astrocytes as peroxynitrite. Thus, spinal cord astrocytes respond to extracellular peroxynitrite by adopting a phenotype that is cytotoxic to motor neurons through peroxynitrite-dependent mechanisms., (Copyright 2002 Wiley-Liss, Inc.)

Amyotrophic lateral sclerosis (ALS) is a lethal disease that is characterized by the relentless death of motoneurons. Mutations to Cu-Zn superoxide dismutase (SOD), though occurring in just 2-3% of individuals with ALS, remain the only proven cause of the disease. These mutations structurally weaken SOD, which indirectly decreases its affinity for Zn. Zn-deficient SOD induces apoptosis in motoneurons through a mechanism involving peroxynitrite. Importantly, Zn-deficient wild-type SOD is just as toxic as Zn-deficient ALS mutant SOD, suggesting that the loss of Zn from wild-type SOD could be involved in the other 98% of cases of ALS. Zn-deficient SOD could therefore be an important therapeutic target in all forms of ALS.

Many of the actions of nitric oxide are not due to nitric oxide itself, but rather by the secondary formation of oxidants like peroxynitrite. Peroxynitrite leaves a footprint in the nitration of tyrosine, which helps track the formation of reactive nitric oxide-derived species in diseases and even normal development.

Inhibition of nitric oxide synthesis prevents rat embryonic motor neurons from undergoing apoptosis when initially cultured without brain-derived neurotrophic factor. Using an improved cell culture medium, we found that the partial withdrawal of trophic support even weeks after motor neurons had differentiated into a mature phenotype still induced apoptosis through a process dependent upon nitric oxide. However, nitric oxide itself was not directly toxic to motor neurons. To investigate whether intracellular superoxide contributed to nitric oxide-dependent apoptosis, we developed a novel method using pH-sensitive liposomes to deliver Cu, Zn superoxide dismutase intracellularly into motor neurons. Intracellular superoxide dismutase prevented motor neuron apoptosis from trophic factor withdrawal, whereas empty liposomes, inactivated superoxide dismutase in liposomes or extracellular superoxide dismutase did not. Neither hydrogen peroxide nor nitrite added separately or in combination affected motor neuron survival. Our results suggest that a partial reduction in trophic support induced motor neuron apoptosis by a process requiring the endogenous production of both nitric oxide and superoxide, irrespective of the extent of motor neuron maturation in culture.

Mutations in copper, zinc superoxide dismutase (SOD) have been implicated in the selective death of motor neurons in 2 percent of amyotrophic lateral sclerosis (ALS) patients. The loss of zinc from either wild-type or ALS-mutant SODs was sufficient to induce apoptosis in cultured motor neurons. Toxicity required that copper be bound to SOD and depended on endogenous production of nitric oxide. When replete with zinc, neither ALS-mutant nor wild-type copper, zinc SODs were toxic, and both protected motor neurons from trophic factor withdrawal. Thus, zinc-deficient SOD may participate in both sporadic and familial ALS by an oxidative mechanism involving nitric oxide.

NO2Tyr (3-Nitrotyrosine) is a modified amino acid that is formed by nitric oxide-derived species and has been implicated in the pathology of diverse human diseases. Nitration of active-site tyrosine residues is known to compromise protein structure and function. Although free NO2Tyr is produced in abundant concentrations under pathological conditions, its capacity to alter protein structure and function at the translational or posttranslational level is unknown. Here, we report that free NO2Tyr is transported into mammalian cells and selectively incorporated into the extreme carboxyl terminus of alpha-tubulin via a posttranslational mechanism catalyzed by the enzyme tubulin-tyrosine ligase. In contrast to the enzymatically regulated carboxyl-terminal tyrosination/detyrosination cycle of alpha-tubulin, incorporation of NO2Tyr shows apparent irreversibility. Nitrotyrosination of alpha-tubulin induces alterations in cell morphology, changes in microtubule organization, loss of epithelial-barrier function, and intracellular redistribution of the motor protein cytoplasmic dynein. These observations imply that posttranslational nitrotyrosination of alpha-tubulin invokes conformational changes, either directly or via allosteric interactions, in the surface-exposed carboxyl terminus of alpha-tubulin that compromises the function of this critical domain in regulating microtubule organization and binding of motor- and microtubule-associated proteins. Collectively, these observations illustrate a mechanism whereby free NO2Tyr can impact deleteriously on cell function under pathological conditions encompassing reactive nitrogen species production. The data also yield further insight into the role that the alpha-tubulin tyrosination/detyrosination cycle plays in microtubule function.

The immunohistochemical detection of nitrotyrosine is a robust method for detecting peroxynitrite and other reactive nitrogen species. Success depends on optimizing conditions for the particular tissue and experimental design under investigation and the use of positive and negative controls to verify specificity. The two controls of dithionite reduction and blocking with nitrotyrosine are a powerful combination to demonstrate specificity. The pathological significance of tyrosine nitration in proteins can also be approached. Generally, nitrated proteins can be isolated from diseased tissues by immunoprecipitation and Western blotting. The sites of nitration on specific proteins can be determined by mass spectrometry, which has revealed surprising specificity in which tyrosines and/or proteins are nitrated in vivo. This provides important evidence concerning the functional consequences of peroxynitrite formation in vivo.

Extracellular trophic factors can regulate whether cells subjected to oxidative stress will survive to proliferate or else undergo cell death. We have previously shown that about 35% of undifferentiated PC12 cells undergo apoptosis 18 h after exposure to peroxynitrite and that pretreatment with nerve growth factor (NGF) protects PC12 cells through activation of phosphatidylinositol (PI) 3-kinase. In contrast, pretreatment with acidic fibroblast growth factor (FGF-1) approximately doubled apoptosis. We report here that NGF added immediately after peroxynitrite treatment no longer protected against apoptosis, but instead enhanced apoptosis to the same extent as FGF. We further investigated which signaling pathways were involved in increasing the level of apoptosis. Overexpression of Bcl-2 blocked the increased apoptosis caused by NGF and FGF-1, but Bcl-2 did not prevent the induction of apoptosis by peroxynitrite alone. The increase in apoptosis caused by the trophic factors was also blocked by the expression of a dominant negative p21Ras mutant. Activation of PI 3-kinase by NGF pretreatment completely protected against both the enhanced apoptosis induced by FGF-1 pretreatment and NGF posttreatment and the apoptosis induced by peroxynitrite alone. Our results indicate that the enhancement of peroxynitrite-induced apoptosis caused by NGF and FGF-1 is dependent on the stimulation of a proapoptotic pathway involving p21Ras that can be suppressed by Bcl-2., (Copyright 1998 Academic Press.)

Trophic factor deprivation induces neuronal nitric oxide synthase (NOS) and apoptosis of rat embryonic motor neurons in culture. We report here that motor neurons constitutively express endothelial NOS that helps support the survival of motor neurons cultured with brain-derived neurotrophic factor (BDNF) by activating the nitric oxide-dependent soluble guanylate cyclase. Exposure of BDNF-treated motor neurons to nitro-L-arginine methyl ester (L-NAME) decreased cell survival 40-50% 24 hr after plating. Both low steady-state concentrations of exogenous nitric oxide (<0.1 microM) and cGMP analogs protected BDNF-treated motor neurons from death induced by L-NAME. Equivalent concentrations of cAMP analogs did not affect cell survival. Inhibition of nitric oxide-sensitive guanylate cyclase with 2 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced the survival of BDNF-treated motor neurons by 35%. cGMP analogs also protected from ODQ-induced motor neuron death, whereas exogenous nitric oxide did not. In all cases, cell death was prevented with caspase inhibitors. Our results suggest that nitric oxide-stimulated cGMP synthesis helps to prevent apoptosis in BDNF-treated motor neurons.

Primary cultures of rat embryonic motor neurons deprived of brain-derived neurotrophic factor (BDNF) induce neuronal nitric oxide synthase (NOS) within 18 hr. Subsequently, >60% of the neurons undergo apoptosis between 18 and 24 hr after plating. Nitro-L-arginine and nitro-L-arginine methyl ester (L-NAME) prevented motor neuron death induced by trophic factor deprivation. Exogenous generation of nitric oxide at concentrations lower than 100 nM overcame the protection by L-NAME. Manganese tetrakis (4-benzoyl acid) porphyrin, a cell-permeant superoxide scavenger, also prevented nitric oxide-dependent motor neuron death. Motor neurons cultured without trophic support rapidly became immunoreactive for nitrotyrosine when compared with motor neurons incubated with BDNF, L-NAME, or manganese TBAP. Our results suggest that peroxynitrite, a strong oxidant formed by the reaction of NO and superoxide, plays an important role in the induction of apoptosis in motor neurons deprived of trophic factors and that BDNF supports motor neuron survival in part by preventing neuronal NOS expression.

Motor neuron survival is highly dependent on trophic factor supply. Deprivation of trophic factors results in induction of neuronal NOS, which is also found in pathological conditions. Growing evidence suggests that motor neuron degeneration involves peroxynitrite formation. Trophic factors modulate peroxynitrite toxicity (Estévez et al., 1995; Shin et al., 1996; Spear et al., 1997). Whether a trophic factor prevents or potentiates peroxynitrite toxicity depends upon when the cells are exposed to the trophic factor (Table 1). These results strongly suggest that a trophic factor that can protect neurons under optimal conditions, but under stressful conditions can increase cell death. In this context, it is possible that trophic factors or cytokines produced as a response to damage may potentiate rather than prevent motor neuron death. A similar argument may apply to the therapeutic administration of trophic factors to treat neurodegenerative diseases. Similarly, the contrasting actions of NO on motor neurons may have important consequences for the potential use of nitric oxide synthase inhibitors in the treatment of ALS and other related neurodegenerative diseases.

Nerve growth factor (NGF) prevents apoptosis induced by the oxidant peroxynitrite in undifferentiated PC12 rat pheochromocytoma cells. Previous studies have shown that activation of phosphatidylinositol 3-kinase (PI 3-kinase) by NGF via the TrkA receptor tyrosine kinase protects PC12 cells from serum deprivation-induced apoptosis. We found that two PI 3-kinase inhibitors, wortmannin and LY294002, eliminated the protection NGF provided against peroxynitrite-induced apoptosis at concentrations consistent with their effectiveness as PI 3-kinase inhibitors. When the activity of PI 3-kinase was assayed in phosphotyrosine immunoprecipitates after treatment of PC12 cells with peroxynitrite, PI 3-kinase activity was reduced by 50% of that detected in control cells, whereas PI 3-kinase activity in NGF-treated cells was unaffected by peroxynitrite. If an antibody against PI 3-kinase was used to immunoprecipitate the enzyme, treatment with peroxynitrite had no effect on activity. Therefore, peroxynitrite appeared to disrupt interactions between PI 3-kinase and phosphotyrosine proteins, rather than directly inhibiting the enzyme. NGF also activates p21Ras-dependent pathways, but this did not appear to be required for NGF to exert its protective effect against peroxynitrite. PC12 cells expressing a dominant inhibitory mutant of p21Ras were equally susceptible to peroxynitrite-induced apoptosis, which was prevented by NGF. Wortmannin was also able to block the protective effect of NGF in the p21Ras mutant cell line. Although many signaling pathways are activated by NGF, these results suggest that a PI 3-kinase-dependent pathway is important for inhibiting peroxynitrite-induced apoptosis.

Peroxynitrite is a powerful oxidant formed by the near-diffusion-limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (> 2 mM) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC50 = 850 microM) intially (3-4 h) caused minimal damage to low-density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite-treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite-treated cells demonstrated DNA fragmentation (by terminal-deoxynucleotide transferase-mediated dUTP-digoxigenin nick end-labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite-induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite-initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite-induced apoptosis in vitro.

Granulosa cells from diethylstilbestrol-treated immature rats were cultured in a defined medium on collagen-coated plates. Thymidine incorporation was significantly increased by insulin (ED50, 656 +/- 110 ng/ml) and insulin-like growth factor (IGF-I; ED50, 95 +/- 10 ng/ml). Insulin and IGF-I stimulations were amplified by methylisobutylxanthine an inhibitor of phosphodiesterase activity. The effect of both peptides were also enhanced by low doses of (Bu)2cAMP (0.2-1 mM). In contrast, higher concentrations were inhibitory. Similarly, FSH produced a biphasic enhancement of the insulin- and IGF-I-stimulated DNA synthesis. Maximal effects (2- to 6-fold increases) were observed with the lower doses (2-20 ng/ml) of the gonadotropin. FSH enhancement of IGF-I-stimulated DNA synthesis was dependent on cell density. Plating densities of 3-5 x 10(5) cells/cm2 were required for a maximal interaction. It is concluded that FSH, acting through a cAMP-mediated pathway, may regulate granulosa cell proliferation by modulating the mitogenic effects of insulin and/or IGF-I.

By using a radioreceptor assay GABA was detectable in rat interscapular brown adipose tissue (IBAT), the levels being 1% those of CNS and 10-fold those of peripheral plasma. Injection of the glutamic acid decarboxylase (GAD) inhibitor 3-mercaptopropionic acid lowered IBAT GABA levels by about half while injection of the GABA transaminase inhibitor gamma-acetylenic GABA increased them by 230%. Rats kept at 4 degrees C for 14 days exhibited IBAT GABA levels that were about half those found at 22 degrees C. Accumulation of IBAT GABA after gamma-acetylenic GABA increased by 2-fold in cold-exposed rats. Sympathetic denervation of IBAT prevented the effect of the cold environment on GABA content and impaired that on GABA accumulation. GAD activity was detectable in IBAT homogenates and isolated brown adipocytes. Exposure of rats to cold increased Vmax of GAD without modifying its Km, regardless of intactness of innervation. In binding studies with 3H-GABA as a ligand, two types of sites were uncovered of KD = 14 and 146 nM, respectively. In the presence of 2.5 mM Ca2+ bicuculline and baclofen were 57 and 46% as effective as GABA to displace 3H-GABA from IBAT binding sites. The results indicate existence, possible synthesis and type A and B receptors of GABA in rat IBAT.

Abstract The objective of the first series of experiments was to assess whether melatonin treatment modifies the activity of the y-aminobutyric acid synthesizing enzyme, glutamic acid decarboxylase, in the preoptic-medial basal hypotnalamic area, cerebral cortex and cerebellar cortex of rats receiving 25 to 300 mug of melatonin in the early morning and late evening in the diurnal cycle. A significant increase of apparent V(max) and K(m) of the enzyme was found in the hypothalamus of rats killed at the 12th h of the light phase (i.e. the time when lights were turned off) and receiving 25 to 300 mug/kg of melatonin 3 h earlier. In the early morning, only a 300 mug/kg dose of melatonin (injected in the 1st h of the light phase) was effective to increase V(max) and K(m) of hypothalamic glutamic acid decarboxylase 3 h later. In cerebral and cerebellar cortices, increases in V(max) and K(m) of enzyme activity were apparent only in the evening and with the highest melatonin dose employed (300 mug/kg). In a second series of experiments the activity of melatonin to modify in vitro(36)CI influx by 900 x g pellets of rat preoptic-medial basal hypothalamic area was studied at the 4th and 12th h of the light phase of daily photoperiod. Melatonin increased (36)CI(-) influx at a minimum concentration of 100 nM (in the morning) or 10 nM (in the evening). The effect of melatonin on (36)CI(-) influx was prevented by co-incubation with 100 muM picrotoxin. Addition of 10 to 100 muM of y-aminobutyric acid to the resuspended 900 x g pellets brought about a dose-dependent increase of (36)CI (-) influx. Preincubation with melatonin at threshold doses of 1 muM (in the morning) or 0.1 muM (in the evening) significantly augmented y-aminobutyric acid effect on (36)CI(-) uptake. These results indicate that melatonin facilitates pre- and postsynaptic activities of y-aminobutyric acid neurons, particularly in the hypothalamus, through an effect that displays a diurnal sensitivity compatible with the documented activity of the hormone on a number of physiological functions.