The putative oxidation of hydroethidine (HE) has become a widely used fluorescent assay for the detection of superoxide in cultured cells. By covalently joining HE to a hexyl triphenylphosphonium cation (Mito-HE), the HE moiety can be targeted to mitochondria. However, the specificity of HE and Mito-HE for superoxide in vivo is limited by autooxidation as well as by nonsuperoxide-dependent cellular processes that can oxidize HE probes to ethidium (Etd).

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 SOD1 G93A rat astrocytes causes astrocytes to induce apoptosis of motor neurons. Mitochondria from SOD1 G93A 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 SOD1 G93A astrocytes, suggesting that mitochondrial defects were associated with nitroxidative damage. Furthermore, superoxide radical formation in mitochondria was increased in SOD1 G93A astrocytes. Similar defects were found in mitochondria isolated from the spinal cord of SOD1 G93A 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, SOD1 G93A 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 SOD1 G93A astrocytes with antioxidants and nitric oxide synthase inhibitors. In particular, low concentrations (ϳ10 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 SOD1 G93A 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.

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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 (p75 NTR ) 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 SOD1 G93A 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 p75 NTR -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 SOD1 G93A astrocytes abolished their apoptotic activity. The protection exerted by increased Nrf2 activity was overcome by adding the NO donor DETANONOate 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. Keywords: amyotrophic lateral sclerosis, astrocytes, glutathione, motor neurons, nuclear factor erythroid 2-related factor 2, p75 neurotrophin receptor. Degenerating neurons can trigger the activation of surrounding astrocytes through the secretion of several proinflammatory mediators. In turn, reactive astrocytes may either promote neuronal survival by providing trophic, metabolic and antioxidant support or stimulate apoptosis by diffusible factors that activate death receptors . Recent evidence showed that mobilization of fibroblast growth factor-1 (FGF-1) can follow motor neuron damage and cause astrocyte activation in an amyotrophic lateral sclerosis (ALS) animal model . Treatment of astrocytes with FGF-1 up-regulates the expression of nerve growth factor (NGF) (Yoshida and Gage 1991), which induces apoptosis of co-cultured motor neurons through activation of the p75 neurotrophin receptor (p75 NTR ) . Although p75NTR is not present in mature motor neurons, the receptor can be re-expressed in pathological conditions, including axotomy (Ernfors et al. 1989;Koliatsos et al. 1991;Rende et al. 1995;Wu 1996;Ferri et al. 1998) Abbreviations used: ALS, amyotrophic lateral sclerosis; ARE, antioxidant response element; BSO, buthionine sulfoximine; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); FGF-1, fibroblast growth factor-1; GFAP, glial fibrillary acidic protein; GSH, reduced glutathione; GSSG, oxidized glutathione; HO-1, heme oxygena...

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.

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.

The import of acetyl-CoA into the ER lumen by AT-1/SLC33A1 is essential for the N -lysine acetylation of ER-resident and ER-transiting proteins. A point-mutation (S113R) in AT-1 has been associated with a familial form of spastic paraplegia. Here, we report that AT-1 S113R is unable to form homodimers in the ER membrane and is devoid of acetyl-CoA transport activity. The reduced influx of acetyl-CoA into the ER lumen results in reduced acetylation of ER proteins and an aberrant form of autophagy. Mice homozygous for the mutation display early developmental arrest. In contrast, heterozygous animals develop to full term, but display neurodegeneration and propensity to infections, inflammation, and cancer. The immune and cancer phenotypes are contingent on the presence of pathogens in the colony, whereas the nervous system phenotype is not. In conclusion, our results reveal a previously unknown aspect of acetyl-CoA metabolism that affects the immune and nervous systems and the risk for malignancies.

Fibroblast growth factor-1 (FGF-1) is highly expressed in motor neurons and can be released in response to sublethal cell injury. Because FGF-1 potently activates astroglia and exerts a direct neuroprotection after spinal cord injury or axotomy, we examined whether it regulated the expression of inducible and cytoprotective heme oxygenase-1 (HO-1) enzyme in astrocytes. FGF-1 induced the expression of HO-1 in cultured rat spinal cord astrocytes, which was dependent on FGF receptor activation and prevented by cycloheximide. FGF-1 also induced Nrf2 mRNA and protein levels and prompted its nuclear translocation. HO-1 induction was abolished by transfection of astrocytes with a dominant-negative mutant Nrf2, indicating that FGF-1 regulates HO-1 expression through Nrf2. FGF-1 also modified the expression of other antioxidant genes regulated by Nrf2. Both Nrf2 and HO-1 levels were increased and co-localized with reactive astrocytes in the degenerating lumbar spinal cord of rats expressing the amyotrophic lateral sclerosis-linked SOD1 G93A mutation. Overexpression of Nrf2 in astrocytes increased survival of co-cultured embryonic motor neurons and prevented motor neuron apoptosis mediated by nerve growth factor through p75 neurotrophin receptor. Taken together, these results emphasize the key role of astrocytes in determining motor neuron fate in amyotrophic lateral sclerosis.Fibroblast growth factor-1 (FGF-1) 1 belongs to a family of polypeptide growth factors (1) that signal through high affinity tyrosine kinase-linked FGF receptors (FGFR1-4) to regulate cell growth, differentiation, and inflammation (2-4). In the spinal cord, FGF-1 is highly expressed in motor neurons where it represents about 0.1% of the soluble protein (5). Because FGF-1 can be released in response to sublethal cell injury (6 -8), FGF-1 mobilization might follow motor neuron damage, causing subsequent autocrine and paracrine effects. FGF-1 has potent direct neurotrophic activity, promotes axonal growth, and exerts beneficial effects in models of spinal cord injury and axon regeneration (9 -13). In the degenerating spinal cord of mice carrying the amyotrophic lateral sclerosis (ALS)-linked Cu,Zn-superoxide dismutase (SOD1) G93A mutation, FGF-1 is up-regulated in the neuropil surrounding motor neurons, whereas astrocytes display FGFR activation (14). FGF-1 is known to stimulate nerve growth factor (NGF) expression and secretion in astrocytes (15), which can promote survival in certain neuronal populations, but paradoxically induce apoptosis of damaged motor neurons expressing p75 neurotrophin receptor (p75 NTR ) (16). Thus, the activation of astrocytes by FGF-1 may have relevant functional consequences for neuronal survival in pathological conditions. Heme oxygenase (HO) is a microsomal enzyme that catalyzes the degradation of heme groups to yield biliverdin, iron, and carbon monoxide (17,18). Biliverdin is subsequently converted to the antioxidant bilirubin by biliverdin reductase. Two isoforms of heme oxygenase have been characterized: a constitutiv...