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BackgroundDissociating glucocorticoid receptor (GR) ligands hold great promise for treating inflammatory disorders since it is assumed that they exert beneficial activities mediated by transrepression but avoid adverse effects of GR action requiring transactivation. Here we challenged this paradigm by investigating 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (CpdA), a dissociating non-steroidal GR ligand, in the context of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).Methodology/Principal FindingsCpdA inhibited pro-inflammatory mediators in myelin-specific T cells and fibroblasts in a GR-dependent manner while gene activation was abolished. However, it also induced massive apoptosis in various cell types even in the absence of the GR by engaging a Bcl-2- and caspase-dependent pathway. 1H NMR spectroscopy corroborated these findings by revealing that CpdA dissolved in buffered solutions rapidly decomposes into aziridine intermediates known to act as alkylating pro-apoptotic agents. Importantly, the dichotomy of CpdA action also became evident in vivo. Administration of high-dose CpdA to mice was lethal while treatment of EAE with low to intermediate amounts of CpdA dissolved in water significantly ameliorated the disease. The beneficial effect of CpdA required expression of the GR in T cells and was achieved by down regulating LFA-1 and CD44 on peripheral Th cells and by repressing IL-17 production.Conclusions/SignificanceCpdA has significant therapeutic potential although adverse effects severely compromise its application in vivo. Hence, non-steroidal GR ligands require careful analysis prior to their translation into new therapeutic concepts.

Rett syndrome (RTT) is a severe neurodevelopmental disorder typically arising from spontaneous mutations in the X-chromosomal methyl-CpG binding protein 2 (MECP2) gene. The almost exclusively female Rett patients show an apparently normal development during their first 6–18 months of life. Subsequently, cognitive- and motor-impairment, hand stereotypies, loss of learned skills, epilepsy and irregular breathing manifest. Early mitochondrial impairment and oxidative challenge are considered to facilitate disease progression. Along this line, we recently confirmed in vitro that acute treatment with the vitamin E-derivative Trolox dampens neuronal hyperexcitability, reinstates synaptic plasticity, ameliorates cellular redox balance and improves hypoxia tolerance in male MeCP2-deficient (Mecp2−/y) mouse hippocampus. Pursuing these promising findings, we performed a preclinical study to define the merit of systemic Trolox administration. Blinded, placebo-controlled in vivo treatment of male mice started at postnatal day (PD) 10–11 and continued for ~40 days. Compounds (vehicle only, 10 mg/kg or 40 mg/kg Trolox) were injected intraperitoneally every 48 h. Detailed phenotyping revealed that in Mecp2−/y mice, blood glucose levels, lipid peroxidation, synaptic short-term plasticity, hypoxia tolerance and certain forms of environmental exploration were improved by Trolox. Yet, body weight and size, motor function and the rate and regularity of breathing did not improve. In conclusion, in vivo Trolox treatment partially ameliorated a subset of symptoms of the complex Rett phenotype, thereby confirming a partial merit of the vitamin E-derivative based pharmacotherapy. Yet, it also became evident that frequent animal handling and the route of drug administration are critical issues to be optimized in future trials.

Rett syndrome (RTT), an X chromosome-linked neurodevelopmental disorder affecting almost exclusively females, is associated with various mitochondrial alterations. Mitochondria are swollen, show altered respiratory rates, and their inner membrane is leaking protons. To advance the understanding of these disturbances and clarify their link to redox impairment and oxidative stress, we assessed mitochondrial respiration in defined brain regions and cardiac tissue of male wildtype (WT) and MeCP2-deficient ( Mecp2 -/y ) mice. Also, we quantified for the first time neuronal redox-balance with subcellular resolution in cytosol and mitochondrial matrix. Quantitative roGFP1 redox imaging revealed more oxidized conditions in the cytosol of Mecp2 -/y hippocampal neurons than in WT neurons. Furthermore, cytosol and mitochondria of Mecp2 -/y neurons showed exaggerated redox-responses to hypoxia and cell-endogenous reactive oxygen species (ROS) formation. Biochemical analyzes exclude disease-related increases in mitochondrial mass in Mecp2 -/y hippocampus and cortex. Protein levels of complex I core constituents were slightly lower in Mecp2 -/y hippocampus and cortex than in WT; those of complex V were lower in Mecp2 -/y cortex. Respiratory supercomplex-formation did not differ among genotypes. Yet, supplied with the complex II substrate succinate, mitochondria of Mecp2 -/y cortex and hippocampus consumed more O 2 than WT. Furthermore, mitochondria from Mecp2 -/y hippocampus and cortex mediated an enhanced oxidative burden. In conclusion, we further advanced the molecular understanding of mitochondrial dysfunction in RTT. Intensified mitochondrial O 2 consumption, increased mitochondrial ROS generation and disturbed redox balance in mitochondria and cytosol may represent a causal chain, which provokes dysregulated proteins, oxidative tissue damage, and contributes to neuronal network dysfunction in RTT.