First described over 50 years ago, Rett syndrome (RTT) is a neurodevelopmental disorder caused primarily by mutations of the X-linked
gene. RTT affects predominantly females, and has a prevalence of ...roughly 1 in every 10,000 female births. Prior to the discovery that mutations of
are the leading cause of RTT, there were suggestions that RTT could be a mitochondrial disease. In fact, several reports documented altered mitochondrial structure, and deficiencies in mitochondrial enzyme activity in different cells or tissues derived from RTT patients. With the identification of
as the causal gene, interest largely shifted toward defining the normal function of MeCP2 in the brain, and how its absence affects the neurodevelopment and neurophysiology. Recently, though, interest in studying mitochondrial function in RTT has been reignited, at least in part due to observations suggesting systemic oxidative stress does play a contributing role in RTT pathogenesis. Here we review data relating to mitochondrial alterations at the structural and functional levels in RTT patients and model systems, and present a hypothesis for how the absence of MeCP2 could lead to altered mitochondrial function and elevated levels of cellular oxidative stress. Finally, we discuss the prospects for treating RTT using interventions that target specific aspects of mitochondrial dysfunction and/or oxidative stress.
Studies on amyloid beta (Aβ|), the peptide thought to play a crucial role in the pathogenesis of Alzheimer's disease, have implicated mitochondria in Aβ-mediated neurotoxicity. We used differentiated ...PC12 cells stably transfected with an inducible green fluorescent protein (GFP) fusion protein containing an N'-terminal mitochondrial targeting sequence (mtGFP), to examine the effects of sub-lethal Aβ on the import of nuclear-encoded proteins to mitochondria. Exposure to sub-lethal Aβ₂₅₋₃₅ (10 μmol/L) for 48 h inhibited mtGFP import to mitochondria; average rates decreased by 20 ± 4%. Concomitant with the decline in mtGFP, cytoplasmic mtGFP increased significantly while mtGFP expression and intramitochondrial mtGFP turnover were unchanged. Sub-lethal Aβ₁₋₄₂ inhibited mtGFP import and increased cytoplasmic mtGFP but only after 96 h. The import of two endogenous nuclear-encoded mitochondrial proteins, mortalin/mtHsp70 and Tom20 also declined. Prior to the decline in import, mitochondrial membrane potential (mmp), and reactive oxygen species levels were unchanged in Aβ-treated cells versus reverse phase controls. Sustained periods of decreased import were associated with decreased mmp, increased reactive oxygen species, increased vulnerability to oxygen-glucose deprivation and altered mitochondrial morphology. These findings suggest that an Aβ-mediated inhibition of mitochondrial protein import, and the consequent mitochondrial impairment, may contribute to Alzheimer's disease.
Abstract Sirt3 is a mitochondrial sirtuin whose deacetylase activity regulates facets of oxidative metabolic efficiency, anti-oxidative capacity, and intra-mitochondrial signaling. In this study, we ...tested whether the over-expression of a human Sirt3-myc transgene in differentiated PC12 cells, a model of sympathetic catecholaminergic neurons, would affect the sensitivity of these cells to oxidative stress or trophic withdrawal insults. Expression analysis revealed the Sirt3-myc product was expressed as a 45 kDa pro-form, which localized primarily within the cytosol, and a 30 kDa processed form that localized predominantly within mitochondria. When subjected to acute glucose deprivation or acute oxygen–glucose deprivation, differentiated PC12 cells over-expressing Sirt3-myc displayed significantly lower levels of cytotoxicity, both at the end of the insult, and at different times following media reperfusion, than cells transfected with a control plasmid. Further, Sirt3-myc over-expression also protected differentiated PC12 cells from apoptosis induced by trophic withdrawal. Collectively, these data indicate that an elevation of Sirt3 is sufficient to protect neuronal PC12 cells from cytotoxic insults, and add to the growing evidence that Sirt3 could be targeted for neuroprotective intervention.
The sirtuins are NAD(+)-dependent protein deacetylases and/or ADP-ribosyltransferases that play roles in metabolic homeostasis, stress response and potentially aging. This enzyme family resides in ...different subcellular compartments, and acts on a number of different targets in the nucleus, cytoplasm and in the mitochondria. Despite their recognized ability to regulate metabolic processes, the roles played by specific sirtuins in the brain-the most energy demanding tissue in the body-remains less well investigated and understood. In the present study, we examined the regional mRNA and protein expression patterns of individual sirtuin family members in the developing, adult, and aged rat brain. Our results show that while each sirtuin is expressed in the brain at each of these different stages, they display unique spatial and temporal expression patterns within the brain. Further, for specific members of the family, the protein expression profile did not coincide with their respective mRNA expression profile. Moreover, using primary cultures enriched for neurons and astrocytes respectively, we found that specific sirtuin members display preferential neural lineage expression. Collectively, these results provide the first composite illustration that sirtuin family members display differential expression patterns in the brain, and provide evidence that specific sirtuins could potentially be targeted to achieve cell-type selective effects within the brain.
Abstract Massive production of free radicals (FR) has been associated with a variety of pathological conditions in the central nervous system (CNS). We have used the FR generating compound hydrogen ...peroxide (H2 O2 ) in organotypic hippocampal slice cultures to model oxidative injury in the brain. Necrotic cell death was monitored for up to 48 h using propidium iodide (PI) and confocal microscopy. A 1 h exposure to H2 O2 (0.5–2.5 mM) caused a dose-dependent, and region specific cell death in hippocampal slice cultures. Glial cells demonstrated a high degree of vulnerability to H2 O2 . During the initial 3 h post-injury period, regions of the slice where glial cell bodies predominated showed massive cell death. The majority of neurons in the pyramidal layers were spared, though at later time points they appeared damaged as well. Carboxy-dichlorofluorescein imaging revealed a corresponding early increase in ROS generation in glial cells compared to pyramidal neurons. Immunohistochemistry of PI labeled slices identified astrocytes as the cells most sensitive to H2 O2 toxicity. In dissociated cell cultures of hippocampal astrocytes and neurons, astrocytes also exhibited a significantly higher sensitivity to H2 O2 than neurons. Hydrogen peroxide-induced cytotoxicity in all regions of the hippocampal slice culture was significantly attenuated by pre-treatment with antioxidants (α-tocopherol and glutathione), and was not prevented by blockade of Ca2+ influx, or NMDA channel activation. Cyclosporin A, an inhibitor of mitochondrial permeability transition, reduced cytotoxicity in glial areas by more than 50%, while in the CA2–CA3 pyramidal layers a much smaller, but still significant, attenuation of cytotoxicity was observed. Our results suggest that mitochondria are primary targets of H2 O2 toxicity, particularly in astrocytes.
Rett syndrome (RTT) is a neurodevelopmental disorder affecting primarily females that is predominantly caused by mutations in the MECP2 gene. RTT is characterized by a loss of previously acquired ...skills, ambulatory deficits, respiratory problems and overall retarded growth. Mitochondrial dysfunction and oxidative stress identified in MeCP2-deficient tissues raised the possibility that mitochondrial impairments may play role in the pathogenesis of RTT. To further investigate the role of mitochondrial dysfunction in the absence of MeCP2, I analyzed mitochondrial function and morphology in Mecp2-deficient mouse adult skin fibroblasts (ASF) and in Mecp2-null mouse ESC derived neurons using an array of fluorescent dyes coupled with flow cytometry and confocal microscopy. The heterogeneity of cellular responses in ASF prevented identification of consistent changes in mitochondrial function, making them an unsuitable model for studying mitochondrial dysfunctions. Mecp2-null mouse ESC were differentiated into enriched population of neurons. Mecp2-null neurons displayed hyperpolarized mitochondria, high levels of ROS, low ATP and impaired mitochondrial trafficking. Resveratrol and mitochondrial cocktail that target expression of mitochondrial genes and mitochondrial metabolism, but not simple ROS scavengers, were successful at ameliorating ROS levels and normalizing mitochondrial membrane potential. Since oxidative stress was reported in RTT mice, I tested whether resveratrol and mitochondrial cocktail could reverse or improve behavioral phenotype in RTT mice. Mitochondrial cocktail but not resveratrol improved exploratory, locomotor and social behavior deficits in RTT mice. Taken together these results suggest that both mitochondrial deficits and oxidative stress are not only the consequences of dysfunctional MeCP2, but are likely the causes of the RTT phenotype. Further, I determined that over-expression of mitochondrial Sirt3, known for its multifaceted effects on mitochondrial metabolism, is sufficient to protect neurons form various cytotoxic insults including oxidative stress. Overall, this study paves the way for potential new therapeutic strategies aimed at improving quality of life in RTT patients.
Background: Neurons swell in response to a variety of insults. The capacity to recover, ie, to shrink, is critical for neuronal function and survival. Studies on dissociated neurons have shown that ...during swelling and shrinking, neurons reorganize their plasma membrane; as neurons swell, in response to hypo-osmotic media, the bilayer area increases. Upon restoration of normo-osmotic media, neurons shrink, forming transient invaginations of the plasma membrane known as vacuole-like dilations (VLDs), to accommodate the decrease in the bilayer. Methods: Here we used confocal microscopy to monitor neuronal swelling and shrinking in the three-dimensional (3D) environment of post-natal rat hippocampal slice cultures. To label neurons, we used biolistic transfection, to introduce enhanced green fluorescent protein (eGFP) targeted to the cytoplasm; and a membrane targeted GFP (lckGFP), targeted to the plasma membrane. Results: Neurons in slice cultures swelled and shrank in response to hypo-osmotic to normo-osmotic media changes. Oxygen-glucose deprivation (OGD) caused sustained neuronal swelling; after reperfusion, some neurons recovered but in others, VLD recovery was stalled. OGD did not impair neuronal capacity to recover from a subsequent osmotic challenge. Conclusion: These results suggest cell surface area regulation (SAR) is an intrinsic property of neurons, and that neuronal capacity for SAR may play an important role in the brain’s response to ischemic insults.
When mitochondrial protein import is inhibited mitochondrial proteins accumulate in cytoplasm. Evidence also implicates proteasome dysfunction and the excessive and/or ectopic accumulation of ...proteins in neurodegenerative diseases. This links proteasome and mitochondrial protein import. Hypotheses: inhibiting mitochondrial protein import causes accumulation of mitochondrial proteins in the cytoplasm and result in proteasome overload causing proteasome dysfunction. Conversely, inhibiting proteasome function and the subsequent accumulation of mitochondrial proteins triggers changes in mitochondrial protein import and activation of retrograde signalling. In PC12 cells sublethal CCCP decreased import of mtGFP and caused its accumulation in cytoplasm creating conditions that favour proteasome overloading Inhibition of proteasome function (MG115) decreased import of mitochondrial proteins and mtGFP in PC12 cells and cortical neurons and caused massive accumulation of mitochondrial proteins in PC12 cells cytoplasm. These results indicate that proteasome inhibition is associated with deficits in mitochondrial protein import and suggest that proteasome dysfunction may disrupt mitochondrial retrograde signalling.
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