Oxidative burden and mitochondrial dysfunction in a mouse model of Rett syndrome.

Rett syndrome is an X chromosome-linked neurodevelopmental disorder associated with cognitive impairment, motor dysfunction and breathing irregularities causing intermittent hypoxia. Evidence for impaired mitochondrial function is also accumulating. A subunit of complex III is among the potentially dys-regulated genes, the inner mitochondrial membrane is leaking protons, brain ATP levels seem reduced, and Rett patient blood samples confirm increased oxidative damage. We therefore screened for mitochondrial dysfunction and impaired redox balance. In hippocampal slices of a Rett mouse model (Mecp2(-/y)) we detected an increased FAD/NADH baseline-ratio indicating intensified oxidization. Cyanide-induced anoxia caused similar decreases in FAD/NADH ratio and mitochondrial membrane potential in both genotypes, but Mecp2(-/y) mitochondria seemed less polarized. Quantifying cytosolic redox balance with the genetically-encoded optical probe roGFP1 confirmed more oxidized baseline conditions, a more vulnerable redox-balance, and more intense responses of Mecp2(-/y) hippocampus to oxidative challenge and mitochondrial impairment. Trolox treatment improved the redox baseline of Mecp2(-/y) hippocampus and dampened its exaggerated responses to oxidative challenge. Microarray analysis of the hippocampal CA1 subfield did not detect alterations of key mitochondrial enzymes or scavenging systems. Yet, quantitative PCR confirmed a moderate upregulation of superoxide dismutase 1 in Mecp2(-/y) hippocampus, which might be a compensatory response to the increased oxidative burden. Since several receptors and ion-channels are redox-modulated, the mitochondrial and redox changes which already manifest in neonates could contribute to the hyperexcitability and diminished synaptic plasticity in MeCP2 deficiency. Therefore, targeting cellular redox balance might qualify as a potential pharmacotherapeutic approach to improve neuronal network function in Rett syndrome.

Oral Feeding of an Antioxidant Cocktail as a Therapeutic Strategy in a Mouse Model of Rett Syndrome: Merits and Limitations of Long-Term Treatment.

Rett syndrome (RTT) is a severe neurodevelopmental disorder that typically arises from spontaneous germline mutations in the X-chromosomal methyl-CpG binding protein 2 (MECP2) gene. For the first 6-18 months of life, the development of the mostly female patients appears normal. Subsequently, cognitive impairment, motor disturbances, hand stereotypies, epilepsy, and irregular breathing manifest, with previously learned skills being lost. Early mitochondrial impairment and a systemic oxidative burden are part of the complex pathogenesis, and contribute to disease progression. Accordingly, partial therapeutic merits of redox-stabilizing and antioxidant (AO) treatments were reported in RTT patients and Mecp2-mutant mice. Pursuing these findings, we conducted a full preclinical trial on male and female mice to define the therapeutic value of an orally administered AO cocktail composed of vitamin E, N-acetylcysteine, and α-lipoic acid. AO treatment ameliorated some of the microcephaly-related aspects. Moreover, the reduced growth, lowered blood glucose levels, and the hippocampal synaptic plasticity of Mecp2-/y mice improved. However, the first-time detected intensified oxidative DNA damage in Mecp2-mutant cortex persisted. The behavioral performance, breathing regularity, and life expectancy of Mecp2-mutant mice did not improve upon AO treatment. Long-term-treated Mecp2+/- mice eventually became obese. In conclusion, the AO cocktail ameliorated a subset of symptoms of the complex RTT-related phenotype, thereby further confirming the potential merits of AO-based pharmacotherapies. Yet, it also became evident that long-term AO treatment may lose efficacy and even aggravate the metabolic disturbances in RTT. This emphasizes the importance of a constantly well-balanced redox balance for systemic well-being.

Systemic Radical Scavenger Treatment of a Mouse Model of Rett Syndrome: Merits and Limitations of the Vitamin E Derivative Trolox.

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.

Redox Indicator Mice Stably Expressing Genetically Encoded Neuronal roGFP: Versatile Tools to Decipher Subcellular Redox Dynamics in Neuropathophysiology.

AIMS: Reactive oxygen species (ROS) and downstream redox alterations not only mediate physiological signaling but also neuropathology. For long, ROS/redox imaging was hampered by a lack of reliable probes. Genetically encoded redox sensors overcame this gap and revolutionized (sub)cellular redox imaging. Yet, the successful delivery of sensor-coding DNA, which demands transfection/transduction of cultured preparations or stereotaxic microinjections of each subject, remains challenging. By generating transgenic mice, we aimed to overcome limiting cultured preparations, circumvent surgical interventions, and to extend effectively redox imaging to complex and adult preparations. RESULTS: Our redox indicator mice widely express Thy1-driven roGFP1 (reduction-oxidation-sensitive green fluorescent protein 1) in neuronal cytosol or mitochondria. Negative phenotypic effects of roGFP1 were excluded and its proper targeting and functionality confirmed. Redox mapping by ratiometric wide-field imaging reveals most oxidizing conditions in CA3 neurons. Furthermore, mitochondria are more oxidized than cytosol. Cytosolic and mitochondrial roGFP1s reliably report cell endogenous redox dynamics upon metabolic challenge or stimulation. Fluorescence lifetime imaging yields stable, but marginal, response ranges. We therefore developed automated excitation ratiometric 2-photon imaging. It offers superior sensitivity, spatial resolution, and response dynamics. INNOVATION AND CONCLUSION: Redox indicator mice enable quantitative analyses of subcellular redox dynamics in a multitude of preparations and at all postnatal stages. This will uncover cell- and compartment-specific cerebral redox signals and their defined alterations during development, maturation, and aging. Cross-breeding with other disease models will reveal molecular details on compartmental redox homeostasis in neuropathology. Combined with ratiometric 2-photon imaging, this will foster our mechanistic understanding of cellular redox signals in their full complexity. Antioxid. Redox Signal. 25, 41-58.

Restraint Stress Intensifies Interstitial K(+) Accumulation during Severe Hypoxia.

Chronic stress affects neuronal networks by inducing dendritic retraction, modifying neuronal excitability and plasticity, and modulating glial cells. To elucidate the functional consequences of chronic stress for the hippocampal network, we submitted adult rats to daily restraint stress for 3 weeks (6 h/day). In acute hippocampal tissue slices of stressed rats, basal synaptic function and short-term plasticity at Schaffer collateral/CA1 neuron synapses were unchanged while long-term potentiation was markedly impaired. The spatiotemporal propagation pattern of hypoxia-induced spreading depression episodes was indistinguishable among control and stress slices. However, the duration of the extracellular direct current potential shift was shortened after stress. Moreover, K(+) fluxes early during hypoxia were more intense, and the postsynaptic recoveries of interstitial K(+) levels and synaptic function were slower. Morphometric analysis of immunohistochemically stained sections suggested hippocampal shrinkage in stressed rats, and the number of cells that are immunoreactive for glial fibrillary acidic protein was increased in the CA1 subfield indicating activation of astrocytes. Western blots showed a marked downregulation of the inwardly rectifying K(+) channel Kir4.1 in stressed rats. Yet, resting membrane potentials, input resistance, and K(+)-induced inward currents in CA1 astrocytes were indistinguishable from controls. These data indicate an intensified interstitial K(+) accumulation during hypoxia in the hippocampus of chronically stressed rats which seems to arise from a reduced interstitial volume fraction rather than impaired glial K(+) buffering. One may speculate that chronic stress aggravates hypoxia-induced pathophysiological processes in the hippocampal network and that this has implications for the ischemic brain.