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Downstream effects of plectin mutations in epidermolysis bullosa simplex with muscular dystrophy
(2016)
Mutations of the human plectin gene (PLEC) on chromosome 8q24 cause autosomal recessive epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). In the present study we analyzed the downstream effects of PLEC mutations on plectin protein expression and localization, the structure of the extrasarcomeric desmin cytoskeleton, protein aggregate formation and mitochondrial distribution in skeletal muscle tissue from three EBS-MD patients.
PLEC gene analysis in a not previously reported 35-year-old EBS-MD patient with additional disease features of cardiomyopathy and malignant arrhythmias revealed novel compound heterozygous (p.(Phe755del) and p.(Lys1040Argfs*139)) mutations resulting in complete abolition of plectin protein expression. In contrast, the other two patients with different homozygous PLEC mutations showed preserved plectin protein expression with one only expressing rodless plectin variants, and the other markedly reduced protein levels. Analysis of skeletal muscle tissue from all three patients revealed severe disruption of the extrasarcomeric intermediate filament cytoskeleton, protein aggregates positive for desmin, syncoilin, and synemin, degenerative myofibrillar changes, and mitochondrial abnormalities comprising respiratory chain dysfunction and an altered organelle distribution and amount.
Our study demonstrates that EBS-MD causing PLEC mutations universally result in a desmin protein aggregate myopathy phenotype despite marked differences in individual plectin protein expression patterns. Since plectin is the key cytolinker protein that regulates the structural and functional organization of desmin filaments, the defective anchorage and spacing of assembled desmin filaments is the key pathogenetic event that triggers the formation of desmin protein aggregates as well as secondary mitochondrial pathology.
Respirasomes are macromolecular assemblies of the respiratory chain complexes I, III and IV in the inner mitochondrial membrane. We determined the structure of supercomplex I1III2IV1 from bovine heart mitochondria by cryo-EM at 9 Å resolution. Most protein-protein contacts between complex I, III and IV in the membrane are mediated by supernumerary subunits. Of the two Rieske iron-sulfur cluster domains in the complex III dimer, one is resolved, indicating that this domain is immobile and unable to transfer electrons. The central position of the active complex III monomer between complex I and IV in the respirasome is optimal for accepting reduced quinone from complex I over a short diffusion distance of 11 nm, and delivering reduced cytochrome c to complex IV. The functional asymmetry of complex III provides strong evidence for directed electron flow from complex I to complex IV through the active complex III monomer in the mammalian supercomplex.
The endoplasmic reticulum–mitochondria encounter structure (ERMES) connects the mitochondrial outer membrane with the ER. Multiple functions have been linked to ERMES, including maintenance of mitochondrial morphology, protein assembly and phospholipid homeostasis. Since the mitochondrial distribution and morphology protein Mdm10 is present in both ERMES and the mitochondrial sorting and assembly machinery (SAM), it is unknown how the ERMES functions are connected on a molecular level. Here we report that conserved surface areas on opposite sides of the Mdm10 β-barrel interact with SAM and ERMES, respectively. We generated point mutants to separate protein assembly (SAM) from morphology and phospholipid homeostasis (ERMES). Our study reveals that the β-barrel channel of Mdm10 serves different functions. Mdm10 promotes the biogenesis of α-helical and β-barrel proteins at SAM and functions as integral membrane anchor of ERMES, demonstrating that SAM-mediated protein assembly is distinct from ER-mitochondria contact sites.
To understand neurodegenerative diseases is one of the major challenges of the 21st century. This also includes Alzheimer´s disease (AD), which represents a chronic neurodegenerative disorder, with long preclinical and prodromal phases (approx. 20 years) and an average clinical duration of 8–10 years. In the early phase of this disease, patients show deterioration of memory, difficulties in finding the right words for everyday objects or mood swings. The risk of AD grows exponentially with age, doubling approximately every 5 to 6 years. AD may contribute to 60–70% of all dementia cases, being the most common cause of this disease. Dementia is one of the major causes of disability and dependency among older people worldwide. The causes of the sporadic form of AD with late onset (LOAD) are not yet known, but it seems to be a result of multiple factors. Neuropathological features are extracellular senile plaques, containing beta-amyloid peptides (Aβ) and intracellular neurofibrillary tangles, containing paired helical tau proteins, which have been associated with neuronal loss and atrophy of the cerebral cortex. Thus, misfolded proteins seem to contribute to the pathogenesis, but are not the only players in the disease process. Developing feasible therapies is difficult due to the multifactorial pathology of AD. Currently approved drugs only attenuate symptoms, but do not cure the disease. Research into AD also has had several failures in terms of developing disease-modifying therapies. Thus, new therapeutic targets in order to develop a causal therapy are desperately needed. Since AD starts many years far before the first symptoms occur, new scientific approaches focus on the early stage, which are discussed to be important in aging and the onset of AD. Today, the hypothesis of the advanced mitochondrial cascade becomes more and more the leading model for LOAD, integrating physiological aging as the main risk factor. Thus, new interventions targeting mitochondrial dysfunction are of substantial interest. Accordingly, the efficacy of Dimebon and TRO19622 to ameliorate mitochondrial dysfunction in cellular and murine models of AD were investigated. Dimebon (Latrepirdine) was, originally developed in Russia as an H1-antiallergic drug. It might specifically interfere with mechanisms relevant for the cognitive decline, especially by improving impaired mitochondrial function and/or dynamics in AD. TRO19622 (Olesoxim) has been identified in a phenotypic screening approach to promote the survival of primary motor neurons. Olesoxim is easily absorbed by cells and accumulates in mitochondria. Olesoxim’s mode of action is not fully understood, however it has been shown to modulate mitochondrial membranes and interact with the voltage-dependent anion channel (VDAC) and the translocator protein (TSPO; also known as PBR). Thereby it inhibits mitochondrial permeability transition. In this study, the effects of Aβ overproduction on mitochondrial function were investigated. The effects of Dimebon and Olesoxim were examined, using a HEK cell line stably transfected with the Swedish APP double mutation (HEKsw) and un-transfected control cells (HEKut). Mitochondrial membrane potential, ATP concentrations, and respirometry were measured. Western Blot analysis of marker proteins for fission & fusion, autophagy, mitogenesis and mPTP formation were performed. Confocal laser scanning microscopy was introduced as a novel method to visualize mitochondrial dynamics. Olesoxim was also tested in Thy-1-C57BJ/6-APPSL mice representing a murine model of AD. For the in vivo model mitochondria from brain tissue were isolated and dissociated brain cells were prepared to determine respiration, lipid peroxidation, MMP, and ATP-levels. Both, the in vitro and in vivo models were compared and discussed in relation to human post-mortem data. The research was conducted in frame of the EU-project entitled „MITOTARGET“ (Mitochondrial dysfunction in neurodegenerative diseases: towards new therapeutics) funded under FP7-Health (http://cordis.europa.eu/result/rcn/54471_en.html). HEKsw cells showed an overall reduction in the mitochondrial respiration, a significant lower MMP, and significantly reduced ATP levels compared to HEKut cells. Mitochondrial mass was equal in both cell lines. In addition most mitochondria in HEKsw cells showed truncated morphology, followed by punctuated mitochondria. Levels of the fission related protein Drp were significantly elevated in HEKsw cells whereas protein levels of fusion related OPA were strongly reduced, leading to a shift in the distribution pattern towards shorter mitochondria. Moreover, HEKsw cells showed reduced mitochondrial density. Protein levels of the translocase of the inner mitochondrial membrane (TIMM50) were strongly diminished in HEKsw cells. The OXPHOS machinery is located in the inner membrane, where the MMP is build up and ATP is generated. Reduced TIMM50 levels in HEKsw indicated a reduction of the inner mitochondrial membrane, which could explain the described deficits in OXPHOS, MMP, ATP and mitochondrial morphology and density. Concentration of both mPTP markers, the voltage-depended anion channel (VDAC) and the peripheral benzodiazepine receptor (PBR), were broadly increased in HEKsw cells. Thy1-APPSL transgenic mice were characterized as in vivo model of AD. Those mice are modified to express the human form of APP, containing both, the Swedish (KM670/671NL) and the London (V717L) double mutations under the murine Thy1 promotor. Beginning at the age of 3 months, Thy1-APPSL mice develop elevated Aβ levels and mitochondrial dysfunction. Mitochondria isolated from brains of Thy-1-C57BJ/6-APPSL mice showed significant impaired respiration, resulting in a reduced MMP. However, ATP levels in dissociated brain cells did not differ compared to controls. Protein levels of FIS were unchanged, whereas Drp levels were significantly increased. Levels of the mitochondrial fusion marker optic atrophie-1 (Opa) protein were significantly reduced. Peroxisome proliferation-activated receptor gamma coactivator 1-alpha (PGC1) is a transcription factor, which represents a master regulator of mitochondrial biogenesis. PGC1 expression was significantly elevated in brains of Thy-1-C57BJ/6-APPSL mice. However, mitochondrial mass seemed to be equal in both mouse lines. Both LC3-Isoforms, the cytosolic and the autophagosomal form, were not changed in brains of Thy-1-C57BJ/6-APPSL mice, which indicates equal mitophagic activity. In brain homogenates, isolated from Thy-1-C57BJ/6-APPSL mice, both mPTP marker, VDAC and PBR, were considerably increased, which is in accordance with the findings in HEKsw cells. In conclusion, both, the cellular (HEKsw) and the animal model of AD (Thy1-APPSL) broadly match pathophysiological features, which have been found in post-mortem samples from AD patients. Thus, HEKsw cells and Thy1-APPSL mice seem to be suitable models to study new treatments against AD. Incubation of HEKsw cells with Dimebon resulted in a remarkable increase in respiratory activity and restored the MMP after impairing the cells with rotenon. Dimebon had no effects on ATP levels in both cell lines, neither after challenging cells with rotenon, nor under basal conditions. By adding Dimebon, citrate synthase (CS) activity in HEKsw cells was increased and mitochondrial morphology was shifted to a tubular shape. Dimebon further enhanced protein levels of Drp and resulted in the compensation of reduced OPA levels. Moreover, Dimebon restored the increased expression levels of the mPTP markers VDAC and PBR. Aβ1-40 levels were significantly decreased in HEKsw cells. However, changes in Aβ1-40 levels seemed to be too small, to solely explain the much larger effects of Dimebon on impaired mitochondrial function. In conclusion, Dimebon treatment restored diverse defects in Aβ overexpressing cells: Aβ levels were reduced, autophagy marker were increased, mitophagy as repair and renewal mechanism was elevated, mitochondrial mass and density were increased, OXPHOS capacity was restored, mitochondrial dynamics were balanced, mitochondrial shape showed a normal distribution, expression levels of the mPTP constituents were reduced, TIMM50 levels augmented to control levels and stress induced MMP and ROS levels were reduced. All these effects were observed after incubation of cells with a rather low concentration of 100 nmol/L. Based on these findings and in addition to already existing literature, Dimebon presents a potential therapeutic option for diseases with accompanied mitochondrial dysfunction. Although, clinical findings published so far are inconsistent. Olesoxim induced a general increase in respiratory activity and enhanced the electron transport (ETS) capacity in HEKsw cells. In addition it normalized the OXPHOS activity almost to control levels. However, incubation using different Olesoxim concentrations led to a dose independent decline in the MMP and decreased ATP levels. Adding Olesoxim caused a dose-dependent change in the length of mitochondria strongly shifting the pattern towards longer mitochondria. In HEKsw cells a reduced mitochondrial density was observed which was reversed by Olesoxim dose-dependently. Olesoxim completely compensated the severely reduced expression levels of TIMM50, but had no effects on TOMM22 levels. An unexpected finding was that 10 µM Olesoxim significantly increased Aβ1-40 levels. Effects of Olesoxim were also tested in vivo. Treatment of Thy-1-C57BJ/6-APPSL mice with Olesoxim restored the impaired MMP in dissociated brain cells, but had no effects on ATP-levels. Olesoxim increased the respiratory activity in isolated brain mitochondria and restored impaired respiration complex activities almost to control levels, without having an effect on CS activity. However, treatment with Olesoxim caused an increase of PGC1 protein levels in brains of Thy-1-C57BJ/6-APPSL mice,beyond basal levels of littermate controls. The mPTP marker proteins voltage-depended anion channel (VDAC) and peripheral benzodiazepine receptor (PBR) were significantly reduced. As well as in the cell models, treatment of Thy-1-C57 BJ/6-APPSL mice with Olesoxim significantly enhanced total human, soluble human and soluble mouse Aβ1-40 levels. Further investigation needs the observation that Olesoxim caused partly negative effects in controls. For instance, Olesoxim reduced the OXPHOS capacity and enhanced protein levels of VADAC and PBR in brains of C57BJ/6 littermate control mice, which could limit the applicability of Olesoxim in further preclinical studies.
Mitochondrial complex I is a 1MDa membrane protein complex with a central role in aerobic energy metabolism. The bioenergetic core functions are executed by 14 central subunits that are conserved from bacteria to man. Despite recent progress in structure determination, our understanding of the function of the ~30 accessory subunits associated with the mitochondrial complex is still limited. We have investigated the structure of complex I from the aerobic yeast Yarrowia lipolytica by cryo-electron microscopy. Our density map at 7.9Å resolution closely matches the 3.6-3.9Å X-ray structure of the Yarrowia lipolytica complex. However, the cryo-EM map indicated an additional subunit on the side of the matrix arm above the membrane surface, pointing away from the membrane arm. The density, which is not present in any previously described complex I structure and occurs in about 20 % of the particles, was identified as the accessory sulfur transferase subunit ST1. The Yarrowia lipolytica complex I preparation is active in generating H2S from the cysteine derivative 3-mercaptopyruvate, catalyzed by ST1. We thus provide evidence for a link between respiratory complex I and mitochondrial sulfur metabolism.
In this meeting report, particularly addressing the topic of protection of the cardiovascular system from ischemia/reperfusion injury, highlights are presented that relate to conditioning strategies of the heart with respect to molecular mechanisms and outcome in patients’ cohorts, the influence of co-morbidities and medications, as well as the contribution of innate immune reactions in cardioprotection. Moreover, developmental or systems biology approaches bear great potential in systematically uncovering unexpected components involved in ischemia–reperfusion injury or heart regeneration. Based on the characterization of particular platelet integrins, mitochondrial redox-linked proteins, or lipid-diol compounds in cardiovascular diseases, their targeting by newly developed theranostics and technologies opens new avenues for diagnosis and therapy of myocardial infarction to improve the patients’ outcome.
Fusion of mitochondrial outer membranes is crucial for proper organelle function and involves large GTPases called mitofusins. The discrete steps that allow mitochondria to attach to one another and merge their outer membranes are unknown. By combining an in vitro mitochondrial fusion assay with electron cryo-tomography (cryo-ET), we visualize the junction between attached mitochondria isolated from Saccharomyces cerevisiae and observe complexes that mediate this attachment. We find that cycles of GTP hydrolysis induce progressive formation of a docking ring structure around extended areas of contact. Further GTP hydrolysis triggers local outer membrane fusion at the periphery of the contact region. These findings unravel key features of mitofusin-dependent fusion of outer membranes and constitute an important advance in our understanding of how mitochondria connect and merge.
Background: NH exchangers (NHEs) play a crucial role in regulating intra/extracellular pH, which is altered in cancer cells, and are therefore suitable targets to alter cancer cell metabolism in order to inhibit cell survival and proliferation. Among NHE inhibitors, amiloride family members are commonly used in clinical practice as diuretics; we focused on the amiloride HMA, reporting a net cytotoxic effect on a panel of human cancer cell lines; now we aim to provide new insights into the molecular events leading to cell death by HMA.
Methods: Colon cancer cell lines were treated with HMA and analysed with: morphological and cellular assays for cell viability and death, and autophagy; biochemical approaches to evaluate mitochondrial function and ROS production; in situ detection of DNA damage; molecular tools to silence crucial autophagy/necroptosis factors.
Results: HMA affects cellular morphology, alters mitochondrial structure and function, causes an increase in ROS, which is detrimental to DNA integrity, stimulates poly(ADP-ribose) synthesis, activates RIPK3-dependent death and triggers autophagy, which is unable to rescue cell survival. These features are hot points of an intricate network of processes, including necroptosis and autophagy, regulating the homeostasis between survival and death.
Conclusion: Our results allow the identification of multiple events leading to cell death in cancer cells treated with HMA. The here-defined intricate network activated by HMA could be instrumental to selectively target the key players of each pathway in the attempt to improve the global response to HMA. Our data could be the starting point for developing a newly designed targeted therapy.