Mechanisms of mitochondria and autophagy crosstalk

Autophagy is a cellular survival pathway that recycles intracellular components to compensate for nutrient depletion and ensures the appropriate degradation of organelles. Mitochondrial number and health are regulated by mitophagy, a process by which excessive or damaged mitochondria are subjected to autophagic degradation. Autophagy is thus a key determinant for mitochondrial health and proper cell function. Mitophagic malfunction has been recently proposed to contribute to progressive neuronal loss in Parkinson disease. In addition to autophagy''s significance in mitochondrial integrity, several lines of evidence suggest that mitochondria can also substantially influence the autophagic process. The mitochondria''s ability to influence and be influenced by autophagy places both elements (mitochondria and autophagy) in a unique position where defects in one or the other system could increase the risk to various metabolic and autophagic related diseases.Key words: autophagy, mitochondria, fission, fusion, apoptosis     

PHYLOGENY OF PHAGOTROPHIC EUGLENIDS (EUGLENOZOA): A MOLECULAR APPROACH BASED ON CULTURE MATERIAL AND ENVIRONMENTAL SAMPLES1

Molecular studies based on small subunit (SSU) rDNA sequences addressing euglenid phylogeny hitherto suffered from the lack of available data about phagotrophic species. To extend the taxon sampling, SSU rRNA genes from species of seven genera of phagotrophic euglenids were investigated. Sequence analyses revealed an increasing genetic diversity among euglenid SSU rDNA sequences compared with other well‐known eukaryotic groups, reflecting an equally broad diversity of morphological characters among euglenid phagotrophs. Phylogenetic inference using standard parsimony and likelihood approaches as well as Bayesian inference and spectral analyses revealed no clear support for euglenid monophyly. Among phagotrophs, monophyly of Petalomonas cantuscygni and Notosolenus ostium, both comprising simple ingestion apparatuses, is strongly supported. A moderately supported clade comprises phototrophic euglenids and primary osmotrophic euglenids together with phagotrophs, exhibiting a primarily flexible pellicle composed of numerous helically arranged strips and a complex ingestion apparatus with two supporting rods and four curved vanes. Comparison of molecular and morphological data is used to demonstrate the difficulties to formulate a hypothesis about how the ingestion apparatus evolved in this group.     

Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases

Intramembrane proteases have the unusual property of cleaving peptide bonds within the lipid bilayer, an environment not obviously suited to a water-requiring hydrolysis reaction. These enzymes include site-2 protease, gamma-secretase/presenilin, signal peptide peptidase and the rhomboids, and they have a wide range of cellular functions. All have multiple transmembrane domains and, because of their high hydrophobicity, have been difficult to purify. We have now developed an in vitro assay to monitor rhomboid activity in the detergent solubilised state. This has allowed us to isolate for the first time a highly pure rhomboid with catalytic activity. Our results suggest that detergent-solubilised rhomboid activity mimics its activity in biological membranes in many aspects. Analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad. This analysis also suggests that other conserved residues participate in subsidiary functions like ligand binding and water supply. We identify a motif shared between rhomboids and the recently discovered derlins, which participate in translocation of misfolded membrane proteins.     

Tuberin – A New Molecular Target in Alzheimer’s Disease?

Tuberous sclerosis complex (TSC) is a common genetic disorder in which affected individuals develop mental retardation, developmental brain defects and seizures. The TSC gene products, hamartin and tuberin, form a complex, of which tuberin is assumed to be the functional component being involved in a wide variety of different cellular processes. Here we report that tuberin protein levels are decreased in the frontal cortex of patients with Alzheimer’s disease. In addition, tuberin levels are also decreased in Down syndrome brain samples positive for β-amyloid plaques and neurofibrillary tangles. Analysis of NeuN revealed that this regulation is not a consequence of differences in the amount of postmitotic neurons. This first connection of tuberin to another common disease beside TSC stimulates new approaches to investigate the molecular development and to establish new therapeutic strategies.     

Lamin A/C-dependent localization of Nesprin-2, a giant scaffolder at the nuclear envelope

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.     

Subfertile couple with t(4;22)(q23;q11.2)

A couple was referred for cytogenetic examination due to idiopathic miscarriages. The proband proved to be a carrier of chromosomal translocation and her partner's karyotype was found to be normal. The karyotype of the proband is 46,XX,t(4;22)(q23;q11.2) and can be regarded as a reason of fertility problems in the investigated couple. The risk of further miscarriages is high, but the risk of a progeny with abnormal karyotype is rather low, as the progeny would probably have lethal imbalances.     

Imaging of ATP membrane transport with dual micro-disk electrodes and scanning electrochemical microscopy

Extracellular adenosine-5'-triphosphate (ATP) is involved in a variety of relevant regulatory mechanisms at a cellular level and has therefore been focus of extensive research. One of the major challenges associated with measuring this key regulatory analyte is the ability to detect and localize extracellular ATP with sufficient spatial and temporal resolution in physiological environments. In this study, scanning electrochemical microscopy (SECM) utilizing an amperometric micro-biosensor based on co-immobilization of the enzymes glucose oxidase and hexokinase is applied for imaging ATP transport through a porous polycarbonate membrane under physiologically relevant conditions. The enzymatic biosensor operates on competitive consumption of the substrate glucose between the immobilized enzymes glucose oxidase and hexokinase involving ATP as a co-substrate. Quantitative determination of the ATP concentration is based on a linear correlation between the glucose consumption and the ATP level. Integration of the amperometric ATP micro-biosensor into a dual micro-disk electrode configuration is achieved by immobilizing the enzymes at one of the micro-disk electrodes while the second disk serves as an unmodified amperometric probe for controlled positioning of the micro-biosensor in close proximity to the sample surface enabling quantification of the obtained current signal.     

Similar mating and sperm displacement patterns in two highly divergent D. simulans populations from Africa and Europe

The frequency of remating in Drosophila melanogaster is affected by both genetic and ecological factors. We studied the remating behaviour in one European (Italy) and one African (Uganda) Drosophila simulans population using six highly polymorphic microsatellite markers. Despite that the populations were genetically distinct (F(ST) = 0.18) and originated from very dissimilar ecological settings with different population densities, we inferred a very similar mating pattern. The remating parameter alpha was similar in both populations (a = 1.3-1.4). No more than two distinct paternal genotypes per family were detected in each population.     

Heterozygous deficiency of manganese superoxide dismutase results in severe lipid peroxidation and spontaneous apoptosis in murine myocardium in vivo

To circumvent the early lethality of manganese superoxide dismutase (SOD2)-deficient mice, we have used a skin-specific strategy with introduction of loxP sites flanking exon 3 of the SOD2 gene. To our surprise, when breeding a female keratin 14 Cre transgenic mouse to a SOD2 "floxed" male mouse, due to keratin 14 promoter-driven Cre expression in the oocytes, all offspring were heterozygous for SOD2. In sharp contrast to initial publications on SOD2(+/-) mice, the herein reported mice on a mixed genetic background (C57BL/6 x 129/Ola) in their heterozygous state (SOD(+/-)) revealed distinct ultrastructural damage of the myocard, with swelling and disruption of mitochondria and accumulation of lipid droplets, increased nitrotyrosine formation, and lipid peroxidation as well as activation of apoptosis signaling pathways in the heart in vivo. Strikingly, and so far unreported, we found a substantial decrease in the activity of the cytosolic copper, zinc superoxide dismutase (SOD1) in the heart tissue of SOD2(+/-) mice, suggesting that the breakdown of mitochondrial membranes in the heart of SOD2(+/-) mice results in the enhanced release of superoxide anion radicals or derivatives thereof with subsequent inactivation of cytosolic SOD1. This model may be particularly suited to long-term studies on age-related heart failure as well as other age-related diseases and the polygenic base of tissue-specific responses to oxidative injury.