Mariana Pehar

Selective fluorescent imaging of superoxide in vivo using ethidium-based probes

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). Recently, superoxide was shown to react with HE to generate 2-hydroxyethidium [Zhao, H., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vasquez-Vivar, J. & Kalyanaraman, B. (2003) Free Radic. Biol. Med. 34, 1359–1368]. However, 2-hydroxyethidium is difficult to distinguish from Etd by conventional fluorescence techniques exciting at 510 nm. While investigating the oxidation of Mito-HE by superoxide, we found that the superoxide product of both HE and Mito-HE could be selectively excited at 396 nm with minimal interference from other nonspecific oxidation products. The oxidation of Mito-HE monitored at 396 nm by antimycin-stimulated mitochondria was 30% slower than at 510 nm, indicating that superoxide production may be overestimated at 510 nm by even a traditional superoxide-stimulating mitochondrial inhibitor. The rate-limiting step for oxidation by superoxide was 4 × 106 M−1·s−1, which is proposed to involve the formation of a radical from Mito-HE. The rapid reaction with a second superoxide anion through radical–radical coupling may explain how Mito-HE and HE can compete for superoxide in vivo with intracellular superoxide dismutases. Monitoring oxidation at both 396 and 510 nm of excitation wavelengths can facilitate the more selective detection of superoxide in vivo.

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.

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.

Altered longevity-assurance activity of p53:p44 in the mouse causes memory loss, neurodegeneration and premature death

The longevity‐assurance activity of the tumor suppressor p53 depends on the levels of Δ40p53 (p44), a short and naturally occurring isoform of the p53 gene. As such, increased dosage of p44 in the mouse leads to accelerated aging and short lifespan. Here we show that mice homozygous for a transgene encoding p44 (p44+/+) display cognitive decline and synaptic impairment early in life. The synaptic deficits are attributed to hyperactivation of insulin‐like growth factor 1 receptor (IGF‐1R) signaling and altered metabolism of the microtubule‐binding protein tau. In fact, they were rescued by either Igf1r or Mapt haploinsufficiency. When expressing a human or a ‘humanized’ form of the amyloid precursor protein (APP), p44+/+ animals developed a selective degeneration of memory‐forming and ‐retrieving areas of the brain, and died prematurely. Mechanistically, the neurodegeneration was caused by both paraptosis‐ and autophagy‐like cell deaths. These results indicate that altered longevity‐assurance activity of p53:p44 causes memory loss and neurodegeneration by affecting IGF‐1R signaling. Importantly, Igf1r haploinsufficiency was also able to correct the synaptic deficits of APP695/swe mice, a model of Alzheimer’s disease.

SLC33A1/AT-1 Protein Regulates the Induction of Autophagy Downstream of IRE1/XBP1 Pathway

Background: AT-1 is an ER membrane transporter that regulates the influx of acetyl-CoA into the ER lumen. Results: IRE1/XBP1 controls the induction of autophagy by regulating AT-1 expression levels and Atg9A acetylation. Conclusion: AT-1 acts downstream of the UPR to control ERAD(II). Significance: Close regulation of the acetylation status of the ER is essential for cell viability during the UPR. One of the main functions of the unfolded protein response is to ensure disposal of large protein aggregates that accumulate in the lumen of the endoplasmic reticulum (ER) whereas avoiding, at least under nonlethal levels of ER stress, cell death. When tightly controlled, autophagy-dependent ER-associated degradation (ERAD(II)) allows the cell to recover from the transient accumulation of protein aggregates; however, when unchecked, it can be detrimental and cause autophagic cell death/type 2 cell death. Here we show that IRE1/XBP1 controls the induction of autophagy/ERAD(II) during the unfolded protein response by activating the ER membrane transporter SLC33A1/AT-1, which ensures continuous supply of acetyl-CoA into the lumen of the ER. Failure to induce AT-1 leads to widespread autophagic cell death. Mechanistically, the regulation of the autophagic process involves Nϵ-lysine acetylation of Atg9A.

AT-1 is the ER membrane acetyl-CoA transporter and is essential for cell viability

The transient or permanent modification of nascent proteins in the early secretory pathway is an essential cellular function that ensures correct folding and maturation of membrane and secreted proteins. We have recently described a new form of post-translational regulation of the membrane protein β-site APP cleaving enzyme 1 (BACE1) involving transient lysine acetylation in the lumen of the endoplasmic reticulum (ER). The essential components of this process are two ER-based acetyl-CoA:lysine acetyltransferases, ATase1 and ATase2, and a membrane transporter that translocates acetyl-CoA into the lumen of the ER. Here, we report the functional identification of acetyl-CoA transporter 1 (AT-1) as the ER membrane acetyl-CoA transporter. We show that AT-1 regulates the acetylation status of ER-transiting proteins, including the membrane proteins BACE1, low-density lipoprotein receptor and amyloid precursor protein (APP). Finally, we show that AT-1 is essential for cell viability as its downregulation results in widespread cell death and induction of features characteristic of autophagy.