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.

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.

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.

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.

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.