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.

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.

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NO 2 Tyr (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 NO 2 Tyr 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 NO 2 Tyr is transported into mammalian cells and selectively incorporated into the extreme carboxyl terminus of ␣-tubulin via a posttranslational mechanism catalyzed by the enzyme tubulin-tyrosine ligase. In contrast to the enzymatically regulated carboxylterminal tyrosination͞detyrosination cycle of ␣-tubulin, incorporation of NO 2 Tyr shows apparent irreversibility. Nitrotyrosination of ␣-tubulin induces alterations in cell morphology, changes in microtubule organization, loss of epithelialbarrier function, and intracellular redistribution of the motor protein cytoplasmic dynein. These observations imply that posttranslational nitrotyrosination of ␣-tubulin invokes conformational changes, either directly or via allosteric interactions, in the surface-exposed carboxyl terminus of ␣-tubulin that compromises the function of this critical domain in regulating microtubule organization and binding of motorand microtubule-associated proteins. Collectively, these observations illustrate a mechanism whereby free NO 2 Tyr 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 ␣-tubulin tyrosination͞detyrosination cycle plays in microtubule function. Nitric oxide (• NO) is a pervasive signaling molecule generated from L-arginine via the catalytic action of both constitutive and inducible forms of • NO synthases (1). A large body of evidence has amassed in the last decade, establishing the operative role of inducible • NO synthase in the pathogenesis of inflammatory, infectious, and degenerative human diseases (2). The detrimental effects ascribed to • NO often arise from its conversion to more reactive species through reactions with partially reduced oxygen species (3).The pathophysiological actions of• NO congeners are primarily rooted in their capacity to alter the function of biological macromolecules through covalent modifications. A metabolite generally reflecting in vivo production of reactive nitrogen intermediates is the amino acid derivative 3-nitrotyrosine (NO 2 Tyr). Evidence for NO 2 Tyr formation in vivo was found when the free amino acid and its deaminated͞ decarboxylated metabolite 3-nitro-4-hydroxyphenylacetic acid were detected as excretory products in human urine (4). The significance of NO 2 Tyr in vivo is highlighted further by observations that protein-linked NO 2 Tyr is markedly elevated in a broad range of human diseases and clinical disorders (5). In vitro studies have identifi...

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.