Patricia Cassina

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.

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.

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.

Extracellular ATP and the P2X7 receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis

BackgroundDuring pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X7 receptors. In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X7 receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1G93A astrocytes.MethodsWe evaluated motor neuron survival after co-culture with SOD1G93A or non-transgenic astrocytes pretreated with agents known to modulate ATP release or P2X7 receptor. We also characterized astrocyte proliferation and extracellular ATP degradation.ResultsRepeated stimulation by ATP or the P2X7-selective agonist BzATP caused astrocytes to become neurotoxic, inducing death of motor neurons. Involvement of P2X7 receptor was further confirmed by Brilliant blue G inhibition of ATP and BzATP effects. In SOD1G93A astrocyte cultures, pharmacological inhibition of P2X7 receptor or increased extracellular ATP degradation with the enzyme apyrase was sufficient to completely abolish their toxicity towards motor neurons. SOD1G93A astrocytes also displayed increased ATP-dependent proliferation and a basal increase in extracellular ATP degradation.ConclusionsHere we found that P2X7 receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death. Remarkably, the neurotoxic phenotype of SOD1G93A astrocytes depended upon basal activation the P2X7 receptor. Thus, pharmacological inhibition of P2X7 receptor might reduce neuroinflammation in ALS through astrocytes.

Phenotypically aberrant astrocytes that promote motoneuron damage in a model of inherited amyotrophic lateral sclerosis

Motoneuron loss and reactive astrocytosis are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a paralytic neurodegenerative disease that can be triggered by mutations in Cu-Zn superoxide dismutase (SOD1). Dysfunctional astrocytes contribute to ALS pathogenesis, inducing motoneuron damage and accelerating disease progression. However, it is unknown whether ALS progression is associated with the appearance of a specific astrocytic phenotype with neurotoxic potential. Here, we report the isolation of astrocytes with aberrant phenotype (referred as “AbA cells”) from primary spinal cord cultures of symptomatic rats expressing the SOD1G93A mutation. Isolation was based on AbA cells’ marked proliferative capacity and lack of replicative senescence, which allowed oligoclonal cell expansion for 1 y. AbA cells displayed astrocytic markers including glial fibrillary acidic protein, S100β protein, glutamine synthase, and connexin 43 but lacked glutamate transporter 1 and the glial progenitor marker NG2 glycoprotein. Notably, AbA cells secreted soluble factors that induced motoneuron death with a 10-fold higher potency than neonatal SOD1G93A astrocytes. AbA-like aberrant astrocytes expressing S100β and connexin 43 but lacking NG2 were identified in nearby motoneurons, and their number increased sharply after disease onset. Thus, AbA cells appear to be an as-yet unknown astrocyte population arising during ALS progression with unprecedented proliferative and neurotoxic capacity and may be potential cellular targets for slowing ALS progression.

Mitochondrial Superoxide Production and Nuclear Factor Erythroid 2-Related Factor 2 Activation in p75 Neurotrophin Receptor-Induced Motor Neuron Apoptosis

Nerve growth factor (NGF) can induce apoptosis by signaling through the p75 neurotrophin receptor (p75NTR) in several nerve cell populations. Cultured embryonic motor neurons expressing p75NTR are not vulnerable to NGF unless they are exposed to an exogenous flux of nitric oxide (•NO). In the present study, we show that p75NTR-mediated apoptosis in motor neurons involved neutral sphingomyelinase activation, increased mitochondrial superoxide production, and cytochrome c release to the cytosol. The mitochondria-targeted antioxidants mitoQ and mitoCP prevented neuronal loss, further evidencing the role of mitochondria in NGF-induced apoptosis. In motor neurons overexpressing the amyotrophic lateral sclerosis (ALS)-linked superoxide dismutase 1G93A (SOD1G93A) mutation, NGF induced apoptosis even in the absence of an external source of •NO. The increased susceptibility of SOD1G93A motor neurons to NGF was associated to decreased nuclear factor erythroid 2-related factor 2 (Nrf2) expression and downregulation of the enzymes involved in glutathione biosynthesis. In agreement, depletion of glutathione in nontransgenic motor neurons reproduced the effect of SOD1G93A expression, increasing their sensitivity to NGF. In contrast, rising antioxidant defenses by Nrf2 activation prevented NGF-induced apoptosis. Together, our data indicate that p75NTR-mediated motor neuron apoptosis involves ceramide-dependent increased mitochondrial superoxide production. This apoptotic pathway is facilitated by the expression of ALS-linked SOD1 mutations and critically modulated by Nrf2 activity.

Complexity of Astrocyte-Motor Neuron Interactions in Amyotrophic Lateral Sclerosis

Neurons and surrounding glial cells compose a highly specialized functional unit. In amyotrophic lateral sclerosis (ALS) astrocytes interact with motor neurons in a complex manner to modulate neuronal survival. Experiments using chimeric mice expressing ALS-linked mutations to Cu,Zn superoxide dismutase (SOD-1) suggest a critical modulation exerted by neighboring non-neuronal cell types on disease phenotype. When perturbed by primary neuronal damage, e.g. expression of SOD-1 mutations, neurons can signal astrocytes to proliferate and become reactive. Fibroblast growth factor-1 (FGF-1) can be released by motor neurons in response to damage to induce astrocyte activation by signaling through the receptor FGFR1. FGF-1 stimulates nerve growth factor (NGF) expression and secretion, as well as activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor. Nrf2 leads to the expression of antioxidant and cytoprotective enzymes such as heme oxygenase-1 and a group of enzymes involved in glutathione metabolism that prevent motor neuron degeneration. However, prolonged stimulation with FGF-1 or SOD-mediated oxidative stress in astrocytes may disrupt the normal neuron-glia interactions and lead to progressive neuronal degeneration. The re-expression of p75 neurotrophin receptor and neuronal NOS in motor neurons in parallel with increased NGF secretion by reactive astrocytes may be a mechanism to eliminate critically damaged neurons. Consequently, astrocyte activation in ALS may have a complex pathogenic role.

PPAR γ activators induce growth arrest and process extension in B12 oligodendrocyte‐like cells and terminal differentiation of cultured oligodendrocytes

Peroxisome proliferator‐activated receptors (PPARs) are key transcription factors in the control of lipid homeostasis and cell differentiation, but little is known about their function in oligodendrocytes, the major lipid‐synthesizing cells in the central nervous system (CNS). Using the B12 oligodendrocyte‐like cell line and rat spinal cord‐derived oligodendrocytes, we evaluated the importance of PPARγ in the maturation process of these cells. B12 cells express all PPAR isoforms (α, β/δ, and γ), as assessed by RT‐PCR, Western‐blot, and transactivation assays. B12 cells respond specifically to PPARγ agonists by arresting cell proliferation and extending cell processes, events that are blocked by the PPARγ antagonist GW9662. In addition, alkyl‐dihydroxyacetone phosphate synthase (ADAPS), a key peroxisomal enzyme involved in the synthesis of myelin‐rich lipid plasmalogens, is increased in PPARγ agonist‐treated B12 cells. In contrast with B12 cells, both immature and mature isolated spinal cord oligodendrocytes presented a high and similar expression level of ADAPS, as assessed by immunocytochemistry. However, as in B12 cells, isolated spinal cord oligodendrocytes were also found to respond specifically to PPARγ agonists with a four‐fold increase in the number of mature cells. Our data suggest a relevant role for PPARγ in oligodendrocyte lipid metabolism and differentiation.