Ferroptosis in health and disease.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.

N1-methylation of adenosine (m1A) in ND5 mRNA leads to complex I dysfunction in Alzheimer's disease.

One mechanism of particular interest to regulate mRNA fate post-transcriptionally is mRNA modification. Especially the extent of m1A mRNA methylation is highly discussed due to methodological differences. However, one single m1A site in mitochondrial ND5 mRNA was unanimously reported by different groups. ND5 is a subunit of complex I of the respiratory chain. It is considered essential for the coupling of oxidation and proton transport. Here we demonstrate that this m1A site might be involved in the pathophysiology of Alzheimer's disease (AD). One of the pathological hallmarks of this neurodegenerative disease is mitochondrial dysfunction, mainly induced by Amyloid ß (Aß). Aß mainly disturbs functions of complex I and IV of the respiratory chain. However, the molecular mechanism of complex I dysfunction is still not fully understood. We found enhanced m1A methylation of ND5 mRNA in an AD cell model as well as in AD patients. Formation of this m1A methylation is catalyzed by increased TRMT10C protein levels, leading to translation repression of ND5. As a consequence, here demonstrated for the first time, TRMT10C induced m1A methylation of ND5 mRNA leads to mitochondrial dysfunction. Our findings suggest that this newly identified mechanism might be involved in Aß-induced mitochondrial dysfunction.

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton.

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

New Probucol Analogues Inhibit Ferroptosis, Improve Mitochondrial Parameters, and Induce Glutathione Peroxidase in HT22 Cells.

Probucol, a hypocholesterolemic compound, is neuroprotective in several models of neurodegenerative diseases but has serious adverse effects in vivo. We now describe the design and synthesis of two new probucol analogues that protect against glutamate-induced oxidative cell death, also known as ferroptosis, in cultured mouse hippocampal (HT22) cells and in primary cortical neurons, while probucol did not show any protective effect. Treatment with both compounds did not affect glutathione depletion but still significantly decreased glutamate-induced production of oxidants, mitochondrial superoxide generation, and mitochondrial hyperpolarization in HT22 cells. Both compounds increase glutathione peroxidase (GPx) 1 levels and GPx activity, also exhibiting protection against RSL3, a GPx4 inactivator. These two compounds are therefore potent activators of GPx activity making further studies of their neuroprotective activity in vivo worthwhile.

Redox Modifications of Proteins of the Mitochondrial Fusion and Fission Machinery.

Mitochondrial fusion and fission tailors the mitochondrial shape to changes in cellular homeostasis. Players of this process are the mitofusins, which regulate fusion of the outer mitochondrial membrane, and the fission protein DRP1. Upon specific stimuli, DRP1 translocates to the mitochondria, where it interacts with its receptors FIS1, MFF, and MID49/51. Another fission factor of clinical relevance is GDAP1. Here, we identify and discuss cysteine residues of these proteins that are conserved in phylogenetically distant organisms and which represent potential sites of posttranslational redox modifications. We reveal that worms and flies possess only a single mitofusin, which in vertebrates diverged into MFN1 and MFN2. All mitofusins contain four conserved cysteines in addition to cysteine 684 in MFN2, a site involved in mitochondrial hyperfusion. DRP1 and FIS1 are also evolutionarily conserved but only DRP1 contains four conserved cysteine residues besides cysteine 644, a specific site of nitrosylation. MFF and MID49/51 are only present in the vertebrate lineage. GDAP1 is missing in the nematode genome and contains no conserved cysteine residues. Our analysis suggests that the function of the evolutionarily oldest proteins of the mitochondrial fusion and fission machinery, the mitofusins and DRP1 but not FIS1, might be altered by redox modifications.

The Charcot-Marie Tooth Disease Mutation R94Q in MFN2 Decreases ATP Production but Increases Mitochondrial Respiration under Conditions of Mild Oxidative Stress.

Charcot-Marie tooth disease is a hereditary polyneuropathy caused by mutations in Mitofusin-2 (MFN2), a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. Autosomal-dominant inheritance of a R94Q mutation in MFN2 causes the axonal subtype 2A2A which is characterized by early onset and progressive atrophy of distal muscles caused by motoneuronal degeneration. Here, we studied mitochondrial shape, respiration, cytosolic, and mitochondrial ATP content as well as mitochondrial quality control in MFN2-deficient fibroblasts stably expressing wildtype or R94Q MFN2. Under normal culture conditions, R94Q cells had slightly more fragmented mitochondria but a similar mitochondrial oxygen consumption, membrane potential, and ATP production as wildtype cells. However, when inducing mild oxidative stress 24 h before analysis using 100 µM hydrogen peroxide, R94Q cells exhibited significantly increased respiration but decreased mitochondrial ATP production. This was accompanied by increased glucose uptake and an up-regulation of hexokinase 1 and pyruvate kinase M2, suggesting increased pyruvate shuttling into mitochondria. Interestingly, these changes coincided with decreased levels of PINK1/Parkin-mediated mitophagy in R94Q cells. We conclude that mitochondria harboring the disease-causing R94Q mutation in MFN2 are more susceptible to oxidative stress, which causes uncoupling of respiration and ATP production possibly by a less efficient mitochondrial quality control.

Dimethyl fumarate alters intracellular Ca2+ handling in immune cells by redox-mediated pleiotropic effects.

Dimethyl fumarate (DMF) is widely used to treat the human autoimmune diseases multiple sclerosis (MS) and psoriasis. DMF causes short-term oxidative stress and activates the antioxidant response via the transcription factor Nrf2 but its immunosuppressive effect is not well understood. Immune cell activation depends on calcium signaling which itself is influenced by the cellular redox state. We therefore measured calcium, reactive oxygen species levels and glutathione content in lymphocytes from immunized mice before onset of experimental autoimmune encephalomyelitis, in peripheral blood mononuclear cells from MS patients treated with DMF, and in mouse splenocytes treated ex vivo with DMF. This demonstrated altered redox states and increased lymphocytic calcium levels in all model systems. DMF caused an immediate influx of calcium from the extracellular space, long-term increased cytosolic calcium levels and reduced calcium stored in intracellular stores. The DMF-elicited current had the electrophysiological characteristics of a transient receptor potential channel and the intracellular calcium levels were normalized by antagonists of TRPA1. Interestingly, the sarco/endoplasmic reticulum Ca2+-ATPase SERCA2b was downregulated but more active due to glutathionylation of the redox-sensitive cysteine 674. DMF therefore causes pleiotropic changes in cellular calcium homeostasis which are likely caused by redox-sensitive post-translational modifications. These changes probably contribute to its immunosuppressive effects.

Crucial role for Nox2 and sleep deprivation in aircraft noise-induced vascular and cerebral oxidative stress, inflammation, and gene regulation.

Aims: Aircraft noise causes endothelial dysfunction, oxidative stress, and inflammation. Transportation noise increases the incidence of coronary artery disease, hypertension, and stroke. The underlying mechanisms are not well understood. Herein, we investigated effects of phagocyte-type NADPH oxidase (Nox2) knockout and different noise protocols (around-the-clock, sleep/awake phase noise) on vascular and cerebral complications in mice. Methods and results: C57BL/6j and Nox2-/- (gp91phox-/-) mice were exposed to aircraft noise (maximum sound level of 85 dB(A), average sound pressure level of 72 dB(A)) around-the-clock or during sleep/awake phases for 1, 2, and 4 days. Adverse effects of around-the-clock noise on the vasculature and brain were mostly prevented by Nox2 deficiency. Around-the-clock aircraft noise of the mice caused the most pronounced vascular effects and dysregulation of Foxo3/circadian clock as revealed by next generation sequencing (NGS), suggesting impaired sleep quality in exposed mice. Accordingly, sleep but not awake phase noise caused increased blood pressure, endothelial dysfunction, increased markers of vascular/systemic oxidative stress, and inflammation. Noise also caused cerebral oxidative stress and inflammation, endothelial and neuronal nitric oxide synthase (e/nNOS) uncoupling, nNOS mRNA and protein down-regulation, and Nox2 activation. NGS revealed similarities in adverse gene regulation between around-the-clock and sleep phase noise. In patients with established coronary artery disease, night-time aircraft noise increased oxidative stress, and inflammation biomarkers in serum. Conclusion: Aircraft noise increases vascular and cerebral oxidative stress via Nox2. Sleep deprivation and/or fragmentation caused by noise triggers vascular dysfunction. Thus, preventive measures that reduce night-time aircraft noise are warranted.

Oxytosis/Ferroptosis-(Re-) Emerging Roles for Oxidative Stress-Dependent Non-apoptotic Cell Death in Diseases of the Central Nervous System.

Although nerve cell death is the hallmark of many neurological diseases, the processes underlying this death are still poorly defined. However, there is a general consensus that neuronal cell death predominantly proceeds by regulated processes. Almost 30 years ago, a cell death pathway eventually named oxytosis was described in neuronal cells that involved glutathione depletion, reactive oxygen species production, lipoxygenase activation, and calcium influx. More recently, a cell death pathway that involved many of the same steps was described in tumor cells and termed ferroptosis due to a dependence on iron. Since then there has been a great deal of discussion in the literature about whether these are two distinct pathways or cell type- and insult-dependent variations on the same pathway. In this review, we compare and contrast in detail the commonalities and distinctions between the two pathways concluding that the molecular pathways involved in the regulation of ferroptosis and oxytosis are highly similar if not identical. Thus, we suggest that oxytosis and ferroptosis should be regarded as two names for the same cell death pathway. In addition, we describe the potential physiological relevance of oxytosis/ferroptosis in multiple neurological diseases.

Presenilin-1 Delta E9 Mutant Induces STIM1-Driven Store-Operated Calcium Channel Hyperactivation in Hippocampal Neurons.

Presenilins regulate calcium homeostasis in the endoplasmic reticulum, and dysregulation of intracellular calcium has been implicated in the pathogenesis of Alzheimer disease. Elevated presenilin-1 (PS1) holoprotein levels have been detected in postmortem brains of patients carrying familial Alzheimer disease (FAD) PS1 mutations. This study examines the effect of the FAD presenilin mutant that lacks the ninth exon (PS1 ∆E9) and does not undergo endoproteolysis on store-operated calcium (SOC) entry. Significant enhancement of SOC channel activation was detected by electrophysiological measurements in hippocampal neurons with PS1 ∆E9 mutant expression. Here, we show that (i) the hyperactivation of SOC channels is mediated by the STIM1 sensor and can be attenuated by STIM1 knockdown or 2-aminoethoxydiphenyl borate application, (ii) the STIM2 is not involved in pathological changes of SOC entry, (iii) the pathological SOC entry demonstrates properties of both TRPC and Orai subunit composition, and (iiii) transgenic Drosophila flies with PS1 ∆E9 expression in the cholinergic neuron system show short-term memory loss, which can be abolished by 2-aminoethoxydiphenyl borate feeding.

The thiol switch C684 in Mitofusin-2 mediates redox-induced alterations of mitochondrial shape and respiration.

Mitofusin-2 (MFN2) is a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. MFN2 also plays a role in mitochondrial fusion induced by changes in the intracellular redox state. Adding oxidized glutathione (GSSG), the core cellular stress indicator, to mitochondrial preparations stimulates mitochondrial fusion by inducing disulphide bond-mediated oligomer formation of MFN2 and its homolog MFN1 which involve cysteine 684 (C684) of MFN2. Mitochondrial hyperfusion represents an adaptive stress response that confers transient protection by increasing mitochondrial ATP production but how this depends on the thiol switch C684 in MFN2 has not been investigated. We now studied mitochondrial function using high-resolution respirometry in cells stably expressing wildtype or C684A MFN2 in MFN2-deficient fibroblasts in response to alterations of the redox state. Empty vector and untransfected cells served as controls. A single treatment of cells with 100 µM hydrogen peroxide 24 h before analysis had no effect on wildtype cells, but normalized the otherwise increased respiration of knockout cells and significantly increased respiration in C684A cells. In line with this, treating permeabilized cells for 10 min with 1 mM GSH greatly reduced respiration only in C684A cells. Our data indicate that mutation of this cysteine which forms disulphide bridges in an oxidative state, apparently renders MFN2 more susceptible to alterations of the redox environment. It remains to be investigated whether other posttranslational modifications like glutathionylation might play an additional role.

Dimethyl fumarate treatment after traumatic brain injury prevents depletion of antioxidative brain glutathione and confers neuroprotection.

Dimethyl fumarate (DMF) is an immunomodulatory compound to treat multiple sclerosis and psoriasis with neuroprotective potential. Its mechanism of action involves activation of the antioxidant pathway regulator Nuclear factor erythroid 2-related factor 2 thereby increasing synthesis of the cellular antioxidant glutathione (GSH). The objective of this study was to investigate whether post-traumatic DMF treatment is beneficial after experimental traumatic brain injury (TBI). Adult C57Bl/6 mice were subjected to controlled cortical impact followed by oral administration of DMF (80 mg/kg body weight) or vehicle at 3, 24, 48, and 72 h after the inflicted TBI. At 4 days after lesion (dal), DMF-treated mice displayed less neurological deficits than vehicle-treated mice and reduced histopathological brain damage. At the same time, the TBI-evoked depletion of brain GSH was prevented by DMF treatment. However, nuclear factor erythroid 2-related factor 2 target gene mRNA expression involved in antioxidant and detoxifying pathways was increased in both treatment groups at 4 dal. Blood brain barrier leakage, as assessed by immunoglobulin G extravasation, inflammatory marker mRNA expression, and CD45+ leukocyte infiltration into the perilesional brain tissue was induced by TBI but not significantly altered by DMF treatment. Collectively, our data demonstrate that post-traumatic DMF treatment improves neurological outcome and reduces brain tissue loss in a clinically relevant model of TBI. Our findings suggest that DMF treatment confers neuroprotection after TBI via preservation of brain GSH levels rather than by modulating neuroinflammation.

Bcl-xL knockout attenuates mitochondrial respiration and causes oxidative stress that is compensated by pentose phosphate pathway activity.

Bcl-xL is an anti-apoptotic protein that localizes to the outer mitochondrial membrane and influences mitochondrial bioenergetics by controlling Ca2+ influx into mitochondria. Here, we analyzed the effect of mitochondrial Bcl-xL on mitochondrial shape and function in knockout (KO), wild type and rescued mouse embryonic fibroblast cell lines. Mitochondria of KO cells were more fragmented, exhibited a reduced ATP concentration, and reduced oxidative phosphorylation (OXPHOS) suggesting an increased importance of ATP generation by other means. Under steady-state conditions, acidification of the growth medium as a readout for glycolysis was similar, but upon inhibition of ATP synthase with oligomycin, KO cells displayed an instant increase in glycolysis. In addition, forced energy production through OXPHOS by replacing glucose with galactose in the growth medium rendered KO cells more susceptible to mitochondrial toxins. KO cells had increased cellular reactive oxygen species and were more susceptible to oxidative stress, but had higher glutathione levels, which were however more rapidly consumed under conditions of oxidative stress. This coincided with an increased activity and protein abundance of the pentose phosphate pathway protein glucose-6-phosphate dehydrogenase, which generates NADPH necessary to regenerate reduced glutathione. KO cells were also less susceptible to pharmacological inhibition of the pentose phosphate pathway. We conclude that mitochondrial Bcl-xL is involved in maintaining mitochondrial respiratory capacity. Its deficiency causes oxidative stress, which is associated with an increased glycolytic capacity and balanced by an increased activity of the pentose phosphate pathway.

Optical coherence tomography for the diagnosis and monitoring of idiopathic intracranial hypertension.

The objectives of the study were to investigate the value of optical coherence tomography in detecting papilledema in patients with idiopathic intracranial hypertension (IIH), a disease which is difficult to monitor and which can lead to permanent visual deficits; to analyze retinal changes over time. In this non-interventional case-control study, spectral-domain optical coherence tomography (SD-OCT) was used to analyze the retinal and optic nerve head (ONH) morphology of 21 patients with IIH and 27 age- and sex-matched healthy controls over time. We analyzed the ONH volume using a custom-made algorithm and employed semi-automated segmentation of macular volume scans to assess the macular retinal nerve fiber layer (RNFL) and ganglion cell layer and inner plexiform layer complex as well as the total macular volume. In IIH patients, the ONH volume was increased and correlated with cerebrospinal fluid (CSF) pressure. The ONH volume decreased after the initiation of treatment with acetazolamide. The macular RNFL volume decreased by 5% in 3.5 months, and a stepwise multivariate regression analysis identified CSF pressure as the main influence on macular RNFL volume at diagnosis. The only factor predicting macular RNFL volume loss over time was ONH volume. SD-OCT can non-invasively monitor changes in retinal and ONH morphology in patients with IIH. Increased ONH volume leads to retinal atrophy in the form of macular RNFL volume loss, presumably due to mechanic jamming of the optic nerve at the disc and subsequent axonal loss.

Absence of system xc- on immune cells invading the central nervous system alleviates experimental autoimmune encephalitis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune demyelinating disease that affects the central nervous system (CNS), leading to neurodegeneration and chronic disability. Accumulating evidence points to a key role for neuroinflammation, oxidative stress, and excitotoxicity in this degenerative process. System xc- or the cystine/glutamate antiporter could tie these pathological mechanisms together: its activity is enhanced by reactive oxygen species and inflammatory stimuli, and its enhancement might lead to the release of toxic amounts of glutamate, thereby triggering excitotoxicity and neurodegeneration. METHODS: Semi-quantitative Western blotting served to study protein expression of xCT, the specific subunit of system xc-, as well as of regulators of xCT transcription, in the normal appearing white matter (NAWM) of MS patients and in the CNS and spleen of mice exposed to experimental autoimmune encephalomyelitis (EAE), an accepted mouse model of MS. We next compared the clinical course of the EAE disease, the extent of demyelination, the infiltration of immune cells and microglial activation in xCT-knockout (xCT-/-) mice and irradiated mice reconstituted in xCT-/- bone marrow (BM), to their proper wild type (xCT+/+) controls. RESULTS: xCT protein expression levels were upregulated in the NAWM of MS patients and in the brain, spinal cord, and spleen of EAE mice. The pathways involved in this upregulation in NAWM of MS patients remain unresolved. Compared to xCT+/+ mice, xCT-/- mice were equally susceptible to EAE, whereas mice transplanted with xCT-/- BM, and as such only exhibiting loss of xCT in their immune cells, were less susceptible to EAE. In none of the above-described conditions, demyelination, microglial activation, or infiltration of immune cells were affected. CONCLUSIONS: Our findings demonstrate enhancement of xCT protein expression in MS pathology and suggest that system xc- on immune cells invading the CNS participates to EAE. Since a total loss of system xc- had no net beneficial effects, these results have important implications for targeting system xc- for treatment of MS.

Dimethyl Fumarate Induces Glutathione Recycling by Upregulation of Glutathione Reductase.

Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate (DMF) is an effective oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. DMF activates the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) leading to increased synthesis of the major cellular antioxidant glutathione (GSH) and prominent neuroprotection in vitro. We previously demonstrated that DMF is capable of raising GSH levels even when glutathione synthesis is inhibited, suggesting enhanced GSH recycling. Here, we found that DMF indeed induces glutathione reductase (GSR), a homodimeric flavoprotein that catalyzes GSSG reduction to GSH by using NADPH as a reducing cofactor. Knockdown of GSR using a pool of E. coli RNase III-digested siRNAs or pharmacological inhibition of GSR, however, also induced the antioxidant response rendering it impossible to verify the suspected attenuation of DMF-mediated neuroprotection. However, in cystine-free medium, where GSH synthesis is abolished, pharmacological inhibition of GSR drastically reduced the effect of DMF on glutathione recycling. We conclude that DMF increases glutathione recycling through induction of glutathione reductase.

Androgen-inducible gene 1 increases the ER Ca(2+) content and cell death susceptibility against oxidative stress.

Androgen-induced gene 1 (AIG1) is a transmembrane protein implicated with survival (its expression level was shown to correlate with the survival of patients suffering from hepatocellular carcinoma) and Ca(2+) signaling (over-expression of AIG1 increased transcription mediated by the Ca(2+)-dependent nuclear factor of activated T cells). We aimed to shed light on this less-studied protein and investigated its tissue expression, genomic organization, intracellular localization and membrane topology as well as its effects on cell death susceptibility and the Ca(2+) content of the endoplasmic reticulum. Immunoblotting of mouse tissues demonstrated highest expression of AIG1 in the liver, lung and heart. AIG1 has a complex genomic organization and expresses several splice variants in a tissue-dependent manner. Analyzing the topology of AIG1 in the ER membrane using a protease-protection assay suggested that AIG has five transmembrane domains with a luminal N- and cytosolic C-terminus and a hydrophobic stretch between the third and fourth membrane domain that does not cross the membrane. AIG1 over-expression slightly increased susceptibility to oxidative stress, which correlated with an increased ER Ca(2+) concentration in two different cell lines. Together, these results indicate that AIG1 plays a role in the control of the intracellular Ca(2+) concentration and cell death susceptibility.

TOX3 regulates neural progenitor identity.

The human genomic locus for the transcription factor TOX3 has been implicated in susceptibility to restless legs syndrome and breast cancer in genome-wide association studies, but the physiological role of TOX3 remains largely unknown. We found Tox3 to be predominantly expressed in the developing mouse brain with a peak at embryonic day E14 where it co-localizes with the neural stem and progenitor markers Nestin and Sox2 in radial glia of the ventricular zone and intermediate progenitors of the subventricular zone. Tox3 is also expressed in neural progenitor cells obtained from the ganglionic eminence of E15 mice that express Nestin, and it specifically binds the Nestin promoter in chromatin immunoprecipitation assays. In line with this, over-expression of Tox3 increased Nestin promoter activity, which was cooperatively enhanced by treatment with the stem cell self-renewal promoting Notch ligand Jagged and repressed by pharmacological inhibition of Notch signaling. Knockdown of Tox3 in the subventricular zone of E12.5 mouse embryos by in utero electroporation of Tox3 shRNA revealed a reduced Nestin expression and decreased proliferation at E14 and a reduced migration to the cortical plate in E16 embryos in electroporated cells. Together, these results argue for a role of Tox3 in the development of the nervous system.

Protein kinase inhibitor ß enhances the constitutive activity of G-protein-coupled zinc receptor GPR39.

GPR39 is a G-protein-coupled zinc receptor that protects against diverse effectors of cell death. Its protective activity is mediated via constitutive activation of Gα13 and the RhoA pathway, leading to increased SRE (serum-response element)-dependent transcription; the zinc-dependent immediate activation of GPR39 involves Gq-mediated increases in cytosolic Ca2+ and Gs coupling leading to increased cAMP levels. We used the cytosolic and soluble C-terminus of GPR39 in a Y2H (yeast-2-hybrid) screen for interacting proteins, thus identifying PKIB (protein kinase A inhibitor ß). Co-expression of GPR39 with PKIB increased the protective activity of GPR39 via the constitutive, but not the ligand-mediated, pathway. PKIB inhibits protein kinase A by direct interaction with its pseudosubstrate domain; mutation of this domain abolished the inhibitory activity of PKIB on protein kinase A activity, but had no effect on the interaction with GPR39, cell protection and induction of SRE-dependent transcription. Zinc caused dissociation of PKIB from GPR39, thereby liberating it to associate with protein kinase A and inhibit its activity, which would result in a negative-feedback loop with the ability to limit activation of the Gs pathway by zinc.

Mitochondrial function and energy metabolism in neuronal HT22 cells resistant to oxidative stress.

BACKGROUND AND PURPOSE: The hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Extracellular glutamate depletes cellular glutathione by blocking the glutamate/cystine antiporter system xc-. Glutathione depletion induces a well-defined programme of cell death characterized by an increase in reactive oxygen species and mitochondrial dysfunction. EXPERIMENTAL APPROACH: We compared the mitochondrial shape, the abundance of mitochondrial complexes and the mitochondrial respiration of HT22 cells, selected based on their resistance to glutamate, with those of the glutamate-sensitive parental cell line. KEY RESULTS: Glutamate-resistant mitochondria were less fragmented and displayed seemingly contradictory features: mitochondrial calcium and superoxide were increased while high-resolution respirometry suggested a reduction in mitochondrial respiration. This was interpreted as a reverse activity of the ATP synthase under oxidative stress, leading to hydrolysis of ATP to maintain or even elevate the mitochondrial membrane potential, suggesting these cells endure ineffective energy metabolism to protect their membrane potential. Glutamate-resistant cells were also resistant to oligomycin, an inhibitor of the ATP synthase, but sensitive to deoxyglucose, an inhibitor of hexokinases. Exchanging glucose with galactose rendered resistant cells 1000-fold more sensitive to oligomycin. These results, together with a strong increase in cytosolic hexokinase 1 and 2, a reduced lactate production and an increased activity of glucose-6-phosphate dehydrogenase, suggest that glutamate-resistant HT22 cells shuttle most available glucose towards the hexose monophosphate shunt to increase glutathione recovery. CONCLUSIONS AND IMPLICATIONS: These results indicate that mitochondrial and metabolic adaptations play an important role in the resistance of cells to oxidative stress.