Increased Mitochondrial Mass and Cytosolic Redox Imbalance in Hippocampal Astrocytes of a Mouse Model of Rett Syndrome: Subcellular Changes Revealed by Ratiometric Imaging of JC-1 and roGFP1 Fluorescence

Abstract

Rett syndrome (RTT) is a neurodevelopmental disorder with mutations in the MECP2 gene. Mostly girls are affected, and an apparently normal development is followed by cognitive impairment, motor dysfunction, epilepsy, and irregular breathing. Various indications suggest mitochondrial dysfunction. In Rett mice, brain ATP levels are reduced, mitochondria are leaking protons, and respiratory complexes are dysregulated. Furthermore, we found in MeCP2-deficient mouse (Mecp2−/y) hippocampus an intensified mitochondrial metabolism and ROS generation. We now used emission ratiometric 2-photon imaging to assess mitochondrial morphology, mass, and membrane potential (ΔΨm) in Mecp2−/y hippocampal astrocytes. Cultured astrocytes were labeled with the ΔΨm marker JC-1, and semiautomated analyses yielded the number of mitochondria per cell, their morphology, and ΔΨm. Mecp2−/y astrocytes contained more mitochondria than wild-type (WT) cells and were more oxidized. Mitochondrial size, ΔΨm, and vulnerability to pharmacological challenge did not differ. The antioxidant Trolox opposed the oxidative burden and decreased the mitochondrial mass, thereby dampening the differences among WT and Mecp2−/y astrocytes; mitochondrial size and ΔΨm were not markedly affected. In conclusion, mitochondrial alterations and redox imbalance in RTT also involve astrocytes. Mitochondria are more numerous in Mecp2−/y than in WT astrocytes. As this genotypic difference is abolished by Trolox, it seems linked to the oxidative stress in RTT.

Neuronal Redox-Imbalance in Rett Syndrome Affects Mitochondria as Well as Cytosol, and Is Accompanied by Intensified Mitochondrial O2 Consumption and ROS Release

“…This is in line with MeCP2-deficient mouse fibroblasts and MECP2 mutant human embryonic stem cells differentiated into neurons (Li et al, 2013; Shulyakova et al, 2017). Yet, we saw a trend toward increased mitochondrial mass in Mecp2 -/y heart, and cell culture studies found higher mitochondrial contents in microglia (Jin et al, 2015) and hippocampal astrocytes of juvenile Rett mice (Bebensee et al, 2017). In part, an increased mitochondrial mass may serve to compensate the mitochondrial underperformance in RTT.…”

“…The cerebral redox-imbalance in RTT is, however, not limited to neurons. Just recently, we found the cytosol of Mecp2 -/y hippocampal astrocytes to be more oxidized than in WTs (Bebensee et al, 2017).…”

“…Subcellular redox conditions were imaged using the genetically encoded roGFP1 sensor (Dooley et al, 2004; Hanson et al, 2004). Whereas our previous studies still relied on lipofection (Großer et al, 2012; Bebensee et al, 2017), we now took advantage of our recently developed viral constructs (Can et al, 2017), to assure a highly neuron-specific expression of roGFP1. With this marked improvement, we specifically addressed redox balance in neurons of Mecp2 -/y hippocampus, and obtained for the very first time a functional readout of redox conditions in living MeCP2-deficient cells at subcellular resolution.…”

Preincubation with a low-dose hydrogen peroxide enhances anti-oxidative stress ability of BMSCs

“…Under physiological conditions, the body continuously produces active oxygen, and the body's antioxidant system continuously removes active oxygen, which is in a dynamic equilibrium, and does not damage the body [19]. However, when harmful stimuli occur, a large amount of active oxygen is generated, and the antioxidant system has limited the ability to remove these, eventually leading to oxidative damage [20]. The main systems for scavenging ROS in cells are antioxidant enzymes such as SOD and CAT, the intracellular levels of both of them can represent the changes of intracellular anti-oxidant capacity to a certain extent.…”

Genetically Encoded Tools for Research of Cell Signaling and Metabolism under Brain Hypoxia

“…Numerous studies have reported the successful application of genetically encoded biosensors that are sensitive to a variety of metabolites and signaling molecules, including ions and neurotransmitters, to investigate the functioning of neurons and glia in in vitro systems under diverse physiological (spontaneous and evoked activity of neuronal and glial cells) or pathological (Parkinson’s disease, Rett syndrome, and spinal muscular atrophy) conditions. Genetically encoded biosensors were applied to visualize the release of neurotransmitters and their intracellular dynamics [ 260 , 261 , 262 ], to observe the metabolic activity (glucose, pyruvate, ATP) and functioning of second messenger systems (Ca 2+ , cAMP) [ 263 , 264 , 265 , 266 ], to detect the formation of ROS [ 267 , 268 , 269 , 270 ], and to record the responses of cellular redox systems [ 183 , 271 , 272 , 273 , 274 , 275 ]. However, many fewer studies have attempted to exploit genetically encoded sensors for the real-time evaluation of alterations in cell signaling and metabolism in in vitro models of brain hypoxic injury.…”