Dynamin-related protein 1 is required for normal mitochondrial bioenergetic and synaptic function in CA1 hippocampal neurons.

Disrupting particular mitochondrial fission and fusion proteins leads to the death of specific neuronal populations; however, the normal functions of mitochondrial fission in neurons are poorly understood, especially in vivo, which limits the understanding of mitochondrial changes in disease. Altered activity of the central mitochondrial fission protein dynamin-related protein 1 (Drp1) may contribute to the pathophysiology of several neurologic diseases. To study Drp1 in a neuronal population affected by Alzheimer's disease (AD), stroke, and seizure disorders, we postnatally deleted Drp1 from CA1 and other forebrain neurons in mice (CamKII-Cre, Drp1lox/lox (Drp1cKO)). Although most CA1 neurons survived for more than 1 year, their synaptic transmission was impaired, and Drp1cKO mice had impaired memory. In Drp1cKO cell bodies, we observed marked mitochondrial swelling but no change in the number of mitochondria in individual synaptic terminals. Using ATP FRET sensors, we found that cultured neurons lacking Drp1 (Drp1KO) could not maintain normal levels of mitochondrial-derived ATP when energy consumption was increased by neural activity. These deficits occurred specifically at the nerve terminal, but not the cell body, and were sufficient to impair synaptic vesicle cycling. Although Drp1KO increased the distance between axonal mitochondria, mitochondrial-derived ATP still decreased similarly in Drp1KO boutons with and without mitochondria. This indicates that mitochondrial-derived ATP is rapidly dispersed in Drp1KO axons, and that the deficits in axonal bioenergetics and function are not caused by regional energy gradients. Instead, loss of Drp1 compromises the intrinsic bioenergetic function of axonal mitochondria, thus revealing a mechanism by which disrupting mitochondrial dynamics can cause dysfunction of axons

Resistance of Dictyostelium discoideum membranes to saponin permeabilization

<p>Abstract</p> <p>Background</p> <p>Saponin is a mild detergent commonly used to permeabilize cells prior to immunofluorescence labeling of intracellular proteins. It has previously been used to that effect in <it>Dictyostelium discoideum </it>amoebae.</p> <p>Findings</p> <p>We show that saponin, contrary to Triton X-100 or alcohol, permeabilizes at best incompletely membranes of <it>Dictyostelium</it>. In cells exposed to osmotic stress, almost complete resistance to saponin permeabilization was observed.</p> <p>Conclusions</p> <p>Saponin should be used with special care to permeabilize <it>Dictyostelium </it>membranes. This unsusual property is presumably linked to the specific sterol composition of <it>Dictyostelium </it>membranes. It may also represent an adaptation of <it>Dictyostelium </it>to harsh conditions or to natural compounds encountered in its natural environment.</p

The Putative Drp1 Inhibitor mdivi-1 Is a Reversible Mitochondrial Complex I Inhibitor that Modulates Reactive Oxygen Species

Mitochondrial fission mediated by the GTPase dynamin-related protein 1 (Drp1) is an attractive drug target in numerous maladies that range from heart disease to neurodegenerative disorders. The compound mdivi-1 is widely reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate brain injury. Here, we show that mdivi-1 reversibly inhibits mitochondrial complex I-dependent O2 consumption and reverse electron transfer-mediated reactive oxygen species (ROS) production at concentrations (e.g., 50 μM) used to target mitochondrial fission. Respiratory inhibition is rescued by bypassing complex I using yeast NADH dehydrogenase Ndi1. Unexpectedly, respiratory impairment by mdivi-1 occurs without mitochondrial elongation, is not mimicked by Drp1 deletion, and is observed in Drp1-deficient fibroblasts. In addition, mdivi-1 poorly inhibits recombinant Drp1 GTPase activity (Ki > 1.2 mM). Overall, these results suggest that mdivi-1 is not a specific Drp1 inhibitor. The ability of mdivi-1 to reversibly inhibit complex I and modify mitochondrial ROS production may contribute to effects observed in disease models. © 2017 Elsevier Inc

DRP1 levels determine the apoptotic threshold during embryonic differentiation through a mitophagy-dependent mechanism.

The changes that drive differentiation facilitate the emergence of abnormal cells that need to be removed before they contribute to further development or the germline. Consequently, in mice in the lead-up to gastrulation, ∼35% of embryonic cells are eliminated. This elimination is caused by hypersensitivity to apoptosis, but how it is regulated is poorly understood. Here, we show that upon exit of naive pluripotency, mouse embryonic stem cells lower their mitochondrial apoptotic threshold, and this increases their sensitivity to cell death. We demonstrate that this enhanced apoptotic response is induced by a decrease in mitochondrial fission due to a reduction in the activity of dynamin-related protein 1 (DRP1). Furthermore, we show that in naive pluripotent cells, DRP1 prevents apoptosis by promoting mitophagy. In contrast, during differentiation, reduced mitophagy levels facilitate apoptosis. Together, these results indicate that during early mammalian development, DRP1 regulation of mitophagy determines the apoptotic response

The Warburg Effect Suppresses Oxidative Stress Induced Apoptosis in a Yeast Model for Cancer

BACKGROUND: Otto Warburg observed that cancer cells are often characterized by intense glycolysis in the presence of oxygen and a concomitant decrease in mitochondrial respiration. Research has mainly focused on a possible connection between increased glycolysis and tumor development whereas decreased respiration has largely been left unattended. Therefore, a causal relation between decreased respiration and tumorigenesis has not been demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: For this purpose, colonies of Saccharomyces cerevisiae, which is suitable for manipulation of mitochondrial respiration and shows mitochondria-mediated cell death, were used as a model. Repression of respiration as well as ROS-scavenging via glutathione inhibited apoptosis and conferred a survival advantage during seeding and early development of this fast proliferating solid cell population. In contrast, enhancement of respiration triggered cell death. CONCLUSION/SIGNIFICANCE: Thus, the Warburg effect might directly contribute to the initiation of cancer formation--not only by enhanced glycolysis--but also via decreased respiration in the presence of oxygen, which suppresses apoptosis

The C. elegans Opa1 Homologue EAT-3 Is Essential for Resistance to Free Radicals

The C. elegans eat-3 gene encodes a mitochondrial dynamin family member homologous to Opa1 in humans and Mgm1 in yeast. We find that mutations in the C. elegans eat-3 locus cause mitochondria to fragment in agreement with the mutant phenotypes observed in yeast and mammalian cells. Electron microscopy shows that the matrices of fragmented mitochondria in eat-3 mutants are divided by inner membrane septae, suggestive of a specific defect in fusion of the mitochondrial inner membrane. In addition, we find that C. elegans eat-3 mutant animals are smaller, grow slower, and have smaller broodsizes than C. elegans mutants with defects in other mitochondrial fission and fusion proteins. Although mammalian Opa1 is antiapoptotic, mutations in the canonical C. elegans cell death genes ced-3 and ced-4 do not suppress the slow growth and small broodsize phenotypes of eat-3 mutants. Instead, the phenotypes of eat-3 mutants are consistent with defects in oxidative phosphorylation. Moreover, eat-3 mutants are hypersensitive to paraquat, which promotes damage by free radicals, and they are sensitive to loss of the mitochondrial superoxide dismutase sod-2. We conclude that free radicals contribute to the pathology of C. elegans eat-3 mutants

Roles of the DYRK Kinase Pom2 in Cytokinesis, Mitochondrial Morphology, and Sporulation in Fission Yeast

Pom2 is predicted to be a dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) related to Pom1 in Schizosaccharomyces pombe. DYRKs share a kinase domain capable of catalyzing autophosphorylation on tyrosine and exogenous phosphorylation on serine/threonine residues. Here we show that Pom2 is functionally different from the well-characterized Pom1, although they share 55% identity in the kinase domain and the Pom2 kinase domain functionally complements that of Pom1. Pom2 localizes to mitochondria throughout the cell cycle and to the contractile ring during late stages of cytokinesis. Overexpression but not deletion of pom2 results in severe defects in cytokinesis, indicating that Pom2 might share an overlapping function with other proteins in regulating cytokinesis. Gain and loss of function analyses reveal that Pom2 is required for maintaining mitochondrial morphology independently of microtubules. Intriguingly, most meiotic pom2Δ cells form aberrant asci with meiotic and/or forespore membrane formation defects. Taken together, Pom2 is a novel DYRK kinase involved in regulating cytokinesis, mitochondrial morphology, meiosis, and sporulation in fission yeast

Vital function of PRELI and essential requirement of its LEA motif

Proteins containing the late embryogenesis abundant (LEA) motif comprise a conserved family, postulated to act as cell protectors. However, their function and mechanisms of action remain unclear. Here we show that PRELI, a mammalian LEA-containing homolog of yeast Ups1p, can associate with dynamin-like GTPase Optic Atrophy-1 (OPA1) and contribute to the maintenance of mitochondrial morphology. Accordingly, PRELI can uphold mitochondrial membrane potential (ΔΨm) and enhance respiratory chain (RC) function, shown by its capacity to induce complex-I/NADH dehydrogenase and ATP synthase expression, increase oxygen consumption and reduce reactive oxygen species (ROS) production. PRELI can also inhibit cell death induced by STS, TNF-α or UV irradiation. Moreover, in vitro and in vivo dominant-negative overexpression of mutant PRELI/LEA− (lacking the LEA motif) and transient in vitro PRELI-specific knockdown can render lymphocytes vulnerable to apoptosis, cause mouse embryo lethality and revert the resistance of lymphoma cells to induced death. Collectively, these data support the long-presumed notion of LEA protein-dependent mechanisms of cytoprotection and suggest that PRELI interacts with OPA1 to maintain mitochondria structures intact, sustain balanced ion−/proton+ gradients, promote oxidative phosphorylation reactions, regulate pro- and antiapoptotic protein traffic and enable cell responses to induced death. These findings may help to understand how bioenergetics is mechanistically connected with cell survival cues

Perturbation of the yeast N-acetyltransferase NatB induces elevation of protein phosphorylation levels

<p>Abstract</p> <p>Background</p> <p>The addition of an acetyl group to protein N-termini is a widespread co-translational modification. NatB is one of the main N-acetyltransferases that targets a subset of proteins possessing an N-terminal methionine, but so far only a handful of substrates have been reported. Using a yeast <it>nat3Δ </it>strain, deficient for the catalytic subunit of NatB, we employed a quantitative proteomics strategy to identify NatB substrates and to characterize downstream effects in <it>nat3Δ</it>.</p> <p>Results</p> <p>Comparing by proteomics WT and <it>nat3Δ </it>strains, using metabolic ¹⁵N isotope labeling, we confidently identified 59 NatB substrates, out of a total of 756 detected acetylated protein N-termini. We acquired in-depth proteome wide measurements of expression levels of about 2580 proteins. Most remarkably, NatB deletion led to a very significant change in protein phosphorylation.</p> <p>Conclusions</p> <p>Protein expression levels change only marginally in between WT and <it>nat3Δ</it>. A comparison of the detected NatB substrates with their orthologous revealed remarkably little conservation throughout the phylogenetic tree. We further present evidence of post-translational N-acetylation on protein variants at non-annotated N-termini. Moreover, analysis of downstream effects in <it>nat3Δ </it>revealed elevated protein phosphorylation levels whereby the kinase Snf1p is likely a key element in this process.</p