Luis Barbeito

Peroxynitrite-induced cytotoxicity in PC12 cells : evidence for an apoptotic mechanism differentially modulated by neurotrophic factors

Abstract: 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 µM) initially (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.

Mitochondrial Dysfunction in SOD1G93A-Bearing Astrocytes Promotes Motor Neuron Degeneration: Prevention by Mitochondrial-Targeted Antioxidants

Mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Recent reports indicate that astrocytes expressing the mutations of superoxide dismutase-1 (SOD1) may contribute to motor neuron injury in ALS. Here, we provide evidence that mitochondrial dysfunction in SOD1G93A rat astrocytes causes astrocytes to induce apoptosis of motor neurons. Mitochondria from SOD1G93A rat astrocytes displayed a defective respiratory function, including decreased oxygen consumption, lack of ADP-dependent respiratory control, and decreased membrane potential. Protein 3-nitrotyrosine was detected immunochemically in mitochondrial proteins from SOD1G93A astrocytes, suggesting that mitochondrial defects were associated with nitroxidative damage. Furthermore, superoxide radical formation in mitochondria was increased in SOD1G93A astrocytes. Similar defects were found in mitochondria isolated from the spinal cord of SOD1G93A rats, and pretreatment of animals with the spin trap 5,5-dimethyl-1-pyrroline N-oxide restored mitochondrial function, forming adducts with mitochondrial proteins in vivo. As shown previously, SOD1G93A astrocytes induced death of motor neurons in cocultures, compared with nontransgenic ones. This behavior was recapitulated when nontransgenic astrocytes were treated with mitochondrial inhibitors. Remarkably, motor neuron loss was prevented by preincubation of SOD1G93A astrocytes with antioxidants and nitric oxide synthase inhibitors. In particular, low concentrations (∼10 nm) of two mitochondrial-targeted antioxidants, ubiquinone and carboxy-proxyl nitroxide, each covalently coupled to a triphenylphosphonium cation (Mito-Q and Mito-CP, respectively), prevented mitochondrial dysfunction, reduced superoxide production in SOD1G93A astrocytes, and restored motor neuron survival. Together, our results indicate that mitochondrial dysfunction in astrocytes critically influences motor neuron survival and support the potential pharmacological utility of mitochondrial-targeted antioxidants in ALS treatment.

Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis.

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 (p75NTR) 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 p75NTR 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 p75NTR +/+ mouse embryos but had no effect in p75NTR –/– 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 p75NTR 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.

Superoxide dismutase and the death of motoneurons in ALS

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.

Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis.

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.

In Vivo Release of Endogenous Amino Acids from the Rat Striatum: Further Evidence for a Role of Glutamate and Aspartate in Corticostriatal Neurotransmission

By means of the push‐pull cannula method, the outflow of endogenous amino acids was studied in the striatum of halothane‐anesthetized rats. Addition of K + ions (30 mM for 4 min) to the superfusion fluid increased the release of aspartate (+116%), glutamate (+ 217%), taurine (+109%), and γ‐aminobutyric acid (GABA) (−429%) whereas a prolonged decrease in the outflow of glutamine (−28%) and a delayed reduction in the efflux of tyrosine (−25%) were observed. In the absence of Ca2‐, the K+‐induced release of aspartate, glutamate, and GABA was blocked whereas the K + ‐induced release of taurine was still present. Under these conditions, the decrease in glutamine efflux was reduced and that of tyrosine was abolished. Local application of tetrodotoxin (5 μM) decreased only the outflow of glutamate (‐25%). One week following lesion of the ipsilateral sensorimotor cortex the spontaneous outflow of glutamine and of tyrosine was enhanced. Despite the lack of change in their spontaneous outflow, the K +‐evoked release of aspartate and glutamate was less pronounced in lesioned than in control animals, whereas the K + ‐evoked changes in GABA and glutamine efflux were not modified. Our data indicate that the push‐pull cannula method is a reliable approach for the study of the in vivo release of endogenous amino acids. In addition, they provide further evidence for a role for glutamate and aspartate as neuro‐transmitters of corticostriatal neurons.

Increased glutathione biosynthesis by Nrf2 activation in astrocytes prevents p75NTR‐dependent motor neuron apoptosis

Astrocytes may modulate the survival of motor neurons in amyotrophic lateral sclerosis (ALS). We have previously shown that fibroblast growth factor‐1 (FGF‐1) activates astrocytes to increase secretion of nerve growth factor (NGF). NGF in turn induces apoptosis in co‐cultured motor neurons expressing the p75 neurotrophin receptor (p75NTR) by a mechanism involving nitric oxide (NO) and peroxynitrite formation. We show here that FGF‐1 increased the expression of inducible nitric oxide synthase and NO production in astrocytes, making adjacent motor neurons vulnerable to NGF‐induced apoptosis. Spinal cord astrocytes isolated from transgenic SOD1G93A rats displayed increased NO production and spontaneously induced apoptosis of co‐cultured motor neurons. FGF‐1 also activates the redox‐sensitive transcription factor nuclear factor erythroid 2‐related factor 2 (Nrf2) in astrocytes. Because Nrf2 increases glutathione (GSH) biosynthesis, we investigated the role of GSH production by astrocytes on p75NTR‐dependent motor neuron apoptosis. The combined treatment of astrocytes with FGF‐1 and t‐butylhydroquinone (tBHQ) increased GSH production and secretion, preventing motor neuron apoptosis. Moreover, Nrf2 activation in SOD1G93A astrocytes abolished their apoptotic activity. The protection exerted by increased Nrf2 activity was overcome by adding the NO donor DETA‐NONOate to the co‐cultures or by inhibiting GSH synthesis and release from astrocytes. These results suggest that activation of Nrf2 in astrocytes can reduce NO‐dependent toxicity to motor neurons by increasing GSH biosynthesis.

FGF-1 induces ATP release from spinal astrocytes in culture and opens pannexin and connexin hemichannels

Spinal astrocytes are coupled by connexin (Cx) gap junctions and express pannexin 1 (Px1) and purinergic receptors. Fibroblast growth factor 1 (FGF-1), which is released in spinal cord injury, activated spinal astrocytes in culture, induced secretion of ATP, and permeabilized them to relatively large fluorescent tracers [ethidium (Etd) and lucifer yellow (LY)] through “hemichannels” (HCs). HCs can be formed by connexins or pannexins; they can open to extracellular space or can form gap junction (GJ) channels, one HC from each cell. (Pannexins may not form gap junctions in mammalian tissues, but they do in invertebrates). HC types were differentiated pharmacologically and by Px1 knockdown with siRNA and by use of astrocytes from Cx43 knockout mice. Permeabilization was reduced by apyrase (APY), an ATPase, and by P2X7 receptor antagonists, implicating secretion of ATP and autocrine and/or paracrine action. Increased permeability of cells exposed to FGF-1 or ATP for 2 h was mediated largely by Px1 HCs activated by P2X7 receptors. After a 7-h treatment, the permeability was mediated by both Cx43 and Px1 HCs. FGF-1 also caused reduction in gap junctional communication. Botulinum neurotoxin A, a blocker of vesicular release, reduced permeabilization when given 30 min before FGF-1 application, but not when given 1 h after FGF-1. We infer that ATP is initially released from vesicles and then it mediates continued release by action on P2X7 receptors and opening of HCs. These changes in HCs and gap junction channels may promote inflammation and deprive neurons of astrocyte-mediated protection in spinal cord trauma and neurodegenerative disease.

Mitochondria in amyotrophic lateral sclerosis: a trigger and a target.

Strong evidence shows that mitochondrial dysfunction is involved in amyotrophic lateral sclerosis (ALS), but despite the fact that mitochondria play a central role in excitotoxicity, oxidative stress and apoptosis, the intimate underlying mechanism linking mitochondrial defects to motor neuron degeneration in ALS still remains elusive. Morphological and functional abnormalities occur in mitochondria in ALS patients and related animal models, although their exact nature and extent are controversial. Recent studies postulate that the mislocalization in mitochondria of mutant forms of copper-zinc superoxide dismutase (SOD1), the only well-documented cause of familial ALS, may account for the toxic gain of function of the enzyme, and hence induce motor neuron death. On the other hand, mitochondrial dysfunction in ALS does not seem to be restricted only to motor neurons as it is also present in other tissues, particularly the skeletal muscle. The presence of this ‘systemic’ defect in energy metabolism associated with the disease is supported in skeletal muscle tissue by impaired mitochondrial respiration and overexpression of uncoupling protein 3. In addition, the lifespan of transgenic mutant SOD1 mice is increased by a highly energetic diet compensating both the metabolic defect and the motorneuronal function. In this review, we will focus on the mitochondrial dysfunction linked to ALS and the cause-and-effect relationships between mitochondria and the pathological mechanisms thought to be involved in the disease.

Chapter 19 Role of endogenous nitric oxide and peroxynitrite formation in the survival and death of motor neurons in culture

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.