Discovery and characterization of selective small molecule inhibitors of the mammalian mitochondrial division dynamin, DRP1.

Balanced rates of mitochondrial division and fusion are required to maintain mitochondrial function, as well as cellular and organismal homeostasis. In mammals, the cellular machines that mediate these processes are dynamin-related GTPases; the cytosolic DRP1 mediates division, while the outer membrane MFN1/2 and inner membrane OPA1 mediate fusion. Unbalanced mitochondrial dynamics are linked to varied pathologies, including cell death and neurodegeneration, raising the possibility that small molecules that target the division and fusion machines to restore balance may have therapeutic potential. Here we describe the discovery of novel small molecules that directly and selectively inhibit assembly-stimulated GTPase activity of the division dynamin, DRP1. In addition, these small molecules restore wild type mtDNA copy number in MFN1 knockout mouse embryonic fibroblast cells, a phenotype linked to deficient mitochondrial fusion activity. Thus, these compounds are unique tools to explore the roles of mitochondrial division in cells, and to assess the potential therapeutic efficacy of rebalancing mitochondrial dynamics in pathologies associated with excessive mitochondrial division.

Inhibition of Drp1 provides neuroprotection in vitro and in vivo.

Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen-glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.

Towards the reconstruction of central nervous system white matter using neural precursor cells.

Epidermal growth factor-responsive neural precursor cells were used as donor cells for transplantation into wild-type and myelin-deficient shiverer (shi) mice. The cells engrafted robustly within the CNS following intracerebroventricular and cisternal transplantation in neonatal mice. The cells adopted glial phenotypes, and some functioned as oligodendrocytes, producing myelin basic protein and morphologically normal internodal myelin sheaths. When individual shi mice received two transplants (on post-natal days 1 and 3), donor-derived cells disseminated widely and expressed myelin basic protein in central white matter tracts throughout the brain.

Scanning electron microscopic analysis of endoscopic versus open vein harvesting techniques.

BACKGROUND: Endoscopic vein harvesting techniques are increasingly used for obtaining conduit for coronary artery bypass surgery. Although they offer advantages in healing over the conventional open technique, concern has been raised regarding the potential for trauma to the vein in the form of intimal disruption which would theoretically predispose to early graft thrombosis and/or development of stenoses. Unfortunately no long term data is yet available for determining if conduits harvested in this fashion are prone to such events. METHODS: We have examined vein segments harvested by both endoscopic and open techniques for evidence of intimal injury (either visible disruption of the intima and/or presence of thrombus) using scanning electron microscopy (SEM). Those harvesting the vein were unaware which patients were in the study, and both the SEM technician and cardiac pathologist who evaluated the scans were blinded to the technique used for harvesting. For each vein segment examined, views were obtained of four different sections and were analyzed at magnifications ranging from 10 yen to 100 yen. RESULTS: Both thrombus formation and visible intimal disruption were identified quite rarely, and overall were not linked significantly to the type of harvesting technique used. CONCLUSIONS: These results suggest that endoscopic vein harvesting techniques do not subject the conduits to more trauma than open techniques and therefore may not predispose to the development of earlier stenoses. This data will need to be confirmed by both other methods of identifying intimal injury and by long-term follow-up of conduit patency in both groups.

Rumpshaker behaves like juvenile-lethal Plp mutations when combined with shiverer in double mutant mice.

The phenotypes of double mutant mice whose genomes are homozygous for an Mbp (myelin basic protein) mutation and hemizygous for a juvenile-lethal Plp (proteolipid protein) mutation were compared in earlier studies. The results suggested that the shiverer Mpb mutation might have some unexplained ability to partially rescue oligodendrocytes (OLs) from the 'death sentence' that is imposed by the Plp mutations. Conversely, they also indicated that the juvenile-lethal Plp mutations may normalize shiverer OL morphology by reducing the numbers of microprocesses. The Plp mutation rumpshaker produces a mild hypomyelination without reduction in OL numbers and a normal lifespan. This report describes double mutant mice combining two Mbp mutations with rumpshaker, utilizing a common B6C3F1 hybrid-based genetic background. Initial studies on B6C3F1 rumpshaker optic nerve and spinal cord white matter showed unanticipated signs of OL death, with morphologic criteria suggestive of an apoptotic mechanism. In shiverer*rumpshaker double mutant mice, this small class of dying cells could not be identified. White matter morphology was similar to that of mice expressing only the shiverer mutation, except that OL microprocesses were far less abundant. This evidence suggests that, despite their distinctive phenotypic differences, rumpshaker may share more characteristics with the juvenile-lethal Plp mutations than has previously been recognized.

Mdv1p is a WD repeat protein that interacts with the dynamin-related GTPase, Dnm1p, to trigger mitochondrial division.

Mitochondrial fission is mediated by the dynamin-related GTPase, Dnm1p, which assembles on the mitochondrial outer membrane into punctate structures associated with sites of membrane constriction and fission. We have identified additional nuclear genes required for mitochondrial fission, termed MDV (for mitochondrial division). MDV1 encodes a predicted soluble protein, containing a coiled-coil motif and seven COOH-terminal WD repeats. Genetic and two-hybrid analyses indicate that Mdv1p interacts with Dnm1p to mediate mitochondrial fission. In addition, Mdv1p colocalizes with Dnm1p in fission-mediating punctate structures on the mitochondrial outer membrane. Whereas localization of Mdv1p to these structures requires Dnm1p, localization of Mdv1p to mitochondrial membranes does not. This indicates that Mdv1p possesses a Dnm1p-independent mitochondrial targeting signal. Dnm1p-independent targeting of Mdv1p to mitochondria requires MDV2. Our data indicate that MDV2 also functions separately to regulate the assembly of Dnm1p into punctate structures. In contrast, Mdv1p is not required for the assembly of Dnm1p, but Dnm1p-containing punctate structures lacking Mdv1p are not able to complete division. Our studies suggest that mitochondrial fission is a multi-step process in which Mdv2p regulates the assembly of Dnm1p into punctate structures and together with Mdv1p functions later during fission to facilitate Dnm1p-dependent mitochondrial membrane constriction and/or division.

The dynamin-related GTPase, Mgm1p, is an intermembrane space protein required for maintenance of fusion competent mitochondria.

Mutations in the dynamin-related GTPase, Mgm1p, have been shown to cause mitochondrial aggregation and mitochondrial DNA loss in Saccharomyces cerevisiae cells, but Mgm1p's exact role in mitochondrial maintenance is unclear. To study the primary function of MGM1, we characterized new temperature sensitive MGM1 alleles. Examination of mitochondrial morphology in mgm1 cells indicates that fragmentation of mitochondrial reticuli is the primary phenotype associated with loss of MGM1 function, with secondary aggregation of mitochondrial fragments. This mgm1 phenotype is identical to that observed in cells with a conditional mutation in FZO1, which encodes a transmembrane GTPase required for mitochondrial fusion, raising the possibility that Mgm1p is also required for fusion. Consistent with this idea, mitochondrial fusion is blocked in mgm1 cells during mating, and deletion of DNM1, which encodes a dynamin-related GTPase required for mitochondrial fission, blocks mitochondrial fragmentation in mgm1 cells. However, in contrast to fzo1 cells, deletion of DNM1 in mgm1 cells restores mitochondrial fusion during mating. This last observation indicates that despite the phenotypic similarities observed between mgm1 and fzo1 cells, MGM1 does not play a direct role in mitochondrial fusion. Although Mgm1p was recently reported to localize to the mitochondrial outer membrane, our studies indicate that Mgm1p is localized to the mitochondrial intermembrane space. Based on our localization data and Mgm1p's structural homology to dynamin, we postulate that it functions in inner membrane remodeling events. In this context, the observed mgm1 phenotypes suggest that inner and outer membrane fission is coupled and that loss of MGM1 function may stimulate Dnm1p-dependent outer membrane fission, resulting in the formation of mitochondrial fragments that are structurally incompetent for fusion.

The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast.

The dynamin-related GTPase Dnm1 controls mitochondrial morphology in yeast. Here we show that dnm1 mutations convert the mitochondrial compartment into a planar 'net' of interconnected tubules. We propose that this net morphology results from a defect in mitochondrial fission. Immunogold labelling localizes Dnm1 to the cytoplasmic face of constricted mitochondrial tubules that appear to be dividing and to the ends of mitochondrial tubules that appear to have recently completed division. The activity of Dnm1 is epistatic to that of Fzo1, a GTPase in the outer mitochondrial membrane that regulates mitochondrial fusion. dnm1 mutations prevent mitochondrial fragmentation in fzo1 mutant strains. These findings indicate that Dnm1 regulates mitochondrial fission, assembling on the cytoplasmic face of mitochondrial tubules at sites at which division will occur.

Lipid deposition in rat aortas with intraluminal hemispherical plug stenosis. A morphological and biophysical study.

A new method was devised to create a stenosis in the rat abdominal aorta. To restrict blood flow, a hemispherical plug was inserted into the aorta through a renal artery. This type of intrinsic (intraluminal) stenosis minimizes possible intramural effects associated with external compression or ligation which severely deform the arterial wall. In the aorta of hypercholesterolemic rats, lipid deposits were distributed in crescent-shaped patches proximal and distal to the plug, whereas lipid deposition in the opposite aortic wall was inhibited. Based on enlarged physical scale models used to study the flow field, the regions of lipid deposition were found to coincide with regions of low shear stress, stagnation, and recirculation. Shear stress was elevated at the wall opposite the plug. These results show that when confounding mural effects are minimized, lipid deposition is promoted in regions of low shear stress with recirculation and inhibited in regions of elevated shear stress.

Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA.

Maintenance of mitochondrial DNA (mtDNA) during cell division is required for progeny to be respiratory competent. Maintenance involves the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid. MGM101 has been identified as a gene essential for mtDNA maintenance in S. cerevisiae, but its role is unknown. Using liquid chromatography coupled with tandem mass spectrometry, we identified Mgm101p as a component of highly enriched nucleoids, suggesting that it plays a nucleoid-specific role in maintenance. Subcellular fractionation, indirect immunofluorescence and GFP tagging show that Mgm101p is exclusively associated with the mitochondrial nucleoid structure in cells. Furthermore, DNA affinity chromatography of nucleoid extracts indicates that Mgm101p binds to DNA, suggesting that its nucleoid localization is in part due to this activity. Phenotypic analysis of cells containing a temperature sensitive mgm101 allele suggests that Mgm101p is not involved in mtDNA packaging, segregation, partitioning or required for ongoing mtDNA replication. We examined Mgm101p's role in mtDNA repair. As compared with wild-type cells, mgm101 cells were more sensitive to mtDNA damage induced by UV irradiation and were hypersensitive to mtDNA damage induced by gamma rays and H2O2 treatment. Thus, we propose that Mgm101p performs an essential function in the repair of oxidatively damaged mtDNA that is required for the maintenance of the mitochondrial genome.

Evidence that CNS hypomyelination does not cause death of jimpy-msd mutant mice.

Mice expressing three of the proteolipid protein (Plp) mutations in the mouse (jimpy, jimpy-msd, and jimpy-4J) all have a severe deficiency of CNS myelin and oligodendrocytes (OLs), and die sometime in their 4th postnatal week. The prevailing view has been that the animals' shortened life span and lack of myelin are causally related. Here we describe the survival of jimpy-msd males for as long as postnatal day (P) 210. Although these spontaneously occurring longer-lived jimpy-msd males show a 2- to 8-fold increase in numbers of myelinated axons in many CNS regions, this does not protect them from a later but still premature death. Investigating the cause of premature death may reveal previously undiscovered properties of the myelin genes or the cells that express them, or perhaps additional unsuspected cellular responses that contribute to the disease. This study identifies small accumulations of inflammatory cells in the brain parenchyma of jimpy-msd mice as young as P14 and as old as P60, suggesting that the pathology of the disease produced by at least this Plp mutation may be far more complex than has been previously recognized.

Quaking*shiverer double-mutant mice survive for at least 100 days with no CNS myelin.

Mice expressing mutations that produce CNS hypomyelination often die prematurely: the more severe the hypomyelination, the shorter the life span. However, we have previously described jimpy-msd mice that survive twice as long as usual; although they acquire significantly increased amounts of myelin, they still succumb long before their unaffected littermates. This result contradicts any postulated causal relationship between extent of CNS hypomyelination and premature death of the animal. Here we have addressed this question in another way, by using an animal model that does not involve a proteolipid protein (Plp) gene mutation. We demonstrate that quaking*shiverer double-mutant mice can survive for at least 100 days without any CNS myelin whatsoever. Therefore, at least for a mouse, absence of CNS myelin is not lethal per se.

Mitochondrial fusion in yeast requires the transmembrane GTPase Fzo1p.

Membrane fusion is required to establish the morphology and cellular distribution of the mitochondrial compartment. In Drosophila, mutations in the fuzzy onions (fzo) GTPase block a developmentally regulated mitochondrial fusion event during spermatogenesis. Here we report that the yeast orthologue of fuzzy onions, Fzo1p, plays a direct and conserved role in mitochondrial fusion. A conditional fzo1 mutation causes the mitochondrial reticulum to fragment and blocks mitochondrial fusion during yeast mating. Fzo1p is a mitochondrial integral membrane protein with its GTPase domain exposed to the cytoplasm. Point mutations that alter conserved residues in the GTPase domain do not affect Fzo1p localization but disrupt mitochondrial fusion. Suborganellar fractionation suggests that Fzo1p spans the outer and is tightly associated with the inner mitochondrial membrane. This topology may be required to coordinate the behavior of the two mitochondrial membranes during the fusion reaction. We propose that the fuzzy onions family of transmembrane GTPases act as molecular switches to regulate a key step in mitochondrial membrane docking and/or fusion.

Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA.

To gain insight into the process of mitochondrial transmission in yeast, we directly labeled mitochondrial proteins and mitochondrial DNA (mtDNA) and observed their fate after the fusion of two cells. To this end, mitochondrial proteins in haploid cells of opposite mating type were labeled with different fluorescent dyes and observed by fluorescence microscopy after mating of the cells. Parental mitochondrial protein markers rapidly redistributed and colocalized throughout zygotes, indicating that during mating, parental mitochondria fuse and their protein contents intermix, consistent with results previously obtained with a single parentally derived protein marker. Analysis of the three-dimensional structure and dynamics of mitochondria in living cells with wide-field fluorescence microscopy indicated that mitochondria form a single dynamic network, whose continuity is maintained by a balanced frequency of fission and fusion events. Thus, the complete mixing of mitochondrial proteins can be explained by the formation of one continuous mitochondrial compartment after mating. In marked contrast to the mixing of parental mitochondrial proteins after fusion, mtDNA (labeled with the thymidine analogue 5-bromodeoxyuridine) remained distinctly localized to one half of the zygotic cell. This observation provides a direct explanation for the genetically observed nonrandom patterns of mtDNA transmission. We propose that anchoring of mtDNA within the organelle is linked to an active segregation mechanism that ensures accurate inheritance of mtDNA along with the organelle.

Introduction of purified alpha 2A-adrenergic receptors into uniformly oriented, unilamellar, phospholipid vesicles: productive coupling to G proteins but lack of receptor-dependent ion transport.

Introduction of highly purified alpha 2A-adrenergic receptors (alpha 2AAR) into lipid vesicles resulted in vesicle preparations that were unilamellar in structure, nonleaky to monovalent cations, and uniformly oriented such that the cytoplasmic domains of the alpha 2AAR faced the vesicle exterior. In this orientation, addition of Gi/G(o) G proteins yielded a 4-5-fold stimulation of agonist-dependent guanosine-5'-O-(3-[35S]thio)triphosphate binding to the G protein alpha subunit. These nonleaky, uniformly oriented, alpha 2AAR-containing vesicle preparations allowed us to explore the hypothesis that the alpha 2AAR itself, or in combination with Gi/G(o) proteins, is able to effect ion translocation. Measurements of 22Na+ uptake, 22Na+ efflux, and H+ movement revealed no detectable agonist-stimulated, receptor-dependent, ion translocation, even in the presence of G proteins, suggesting that allosteric regulation of alpha 2AAR by cations and amiloride analogs is not an indication that the alpha 2AAR itself is an ion transporter. Nonetheless, the methodology developed in the present studies for preparation of nonleaky vesicles containing receptor and G proteins should be well suited for evaluating the stoichiometry and selectivity of receptor-G protein interactions and, in particular, G protein specificity in mediating receptor-dependent regulation of voltage-gated or receptor-operated ion channels.

A mitochondrial protease with two catalytic subunits of nonoverlapping specificities.

The mitochondrial inner membrane protease is required for the maturation of mitochondrial proteins that are delivered to the intermembrane space. In the yeast Saccharomyces cerevisiae, this protease is now shown to be a complex that contains two catalytic subunits, Imp2p and the previously identified Imp1p. Primary structure similarity indicates that Imp1p and Imp2p are related to each other and to the family of eubacterial and eukaryotic signal peptidases. Imp1p and Imp2p have separate, nonoverlapping substrate specificities. In addition to its catalyzing the cleavage of intermembrane space sorting signals, Imp2p is required for the stable and functional expression of Imp1p. Thus, inner membrane protease, and by analogy eukaryotic multisubunit signal peptidases, may have acquired multiple catalytic subunits by gene duplication to broaden their range of substrate specificity.

Inhibition of lipid deposition in the hypercholesterolemic rat by clentiazem, a calcium channel blocker.

We studied the effects of clentiazem, a calcium channel blocker (1) on the accumulation of lipid in the aorta, (2) on the level of plasma lipids, and (3) on the number of adherent intimal monocytes and foam cells. Seventy Wistar rats were assigned to one of the following groups: (1) regular diet, (2) an atherogenic diet consisting of regular chow with 2% cholesterol, 1% cholic acid, and 0.5% thiouracil (CCT), (3) CCT supplemented with 5 mg/kg/day clentiazem, and (4) CCT with 25 mg/kg/day clentiazem. Animals were sacrificed after 6 or 12 weeks of diet. Aortas were studied by light microscopy after staining with oil red O (ORO) and/or hematoxylin. ORO staining was quantified in both abdominal and thoracic regions of the aorta. The aortas of the clentiazem groups demonstrated significantly less ORO staining than CCT diet controls in thoracic aorta after 6 weeks and abdominal aorta after 12 weeks. There was no significant difference in the plasma lipid concentrations. The clentiazem-treated groups had fewer numbers of adherent monocytes and foam cells. We conclude that clentiazem inhibits lipid deposition in cholesterol-fed rats without lowering plasma lipid concentrations and that the number of intimal monocytes and foam cells is decreased in the presence of this calcium antagonist.

Protein targeting to and translocation across the membrane of the endoplasmic reticulum.

Several approaches are currently being taken to elucidate the mechanisms and the molecular components responsible for protein targeting to and translocation across the membrane of the endoplasmic reticulum. Two experimental systems dominate the field: a biochemical system derived from mammalian exocrine pancreas, and a combined genetic and biochemical system employing the yeast, Saccharomyces cerevisiae. Results obtained in each of these systems have contributed novel, mostly non-overlapping information. Recently, much effort in the field has been dedicated to identifying membrane proteins that comprise the translocon. Membrane proteins involved in translocation have been identified both in the mammalian system, using a combination of crosslinking and reconstitution approaches, and in S. cerevisiae, by selecting for mutants in the translocation pathway. None of the membrane proteins isolated, however, appears to be homologous between the two experimental systems. In the case of the signal recognition particle, the two systems have converged, which has led to a better understanding of how proteins are targeted to the endoplasmic reticulum membrane.

Doxazosin and cholestyramine similarly decrease fatty streak formation in the aortic arch of hyperlipidemic hamsters.

The effect of doxazosin, an alpha-1 adrenergic inhibitor, on atherosclerosis was determined in hyperlipidemic hamsters. Control hamsters fed chow plus 0.05% cholesterol and 10% coconut oil were compared to chow baseline animals, and to those receiving either 10 mg/kg/day doxazosin, or 245 mg/kg/day cholestyramine in the atherogenic diet. During 8 weeks of treatment, plasma lipids, mean arterial pressure (MAP) and heart rate (HR) were measured, then the ascending aortic arch was examined en face. Numbers of subendothelial macrophage-foam cells/mm2 and their average size (microns 2) were determined, and Oil red O staining (micrograms ORO/mm2) was quantitated to estimate lipid accumulation. Ultracentrifugation of control plasma demonstrate that low density lipoprotein (LDL) carried most of the cholesterol, and very low density lipoprotein (VLDL) was rich in triglycerides. Compared to controls, doxazosin and cholestyramine similarly decreased plasma total and LDL plus VLDL cholesterols, and total triglycerides on average by 46%, 61% and 45% respectively. High density lipoprotein cholesterol was unchanged. Doxazosin also reduced MAP by 18% without affecting HR. In all hamsters, foam cells and lipid accumulated in a lesion-prone area characterized by elevated endothelial cell density, and a thick intima of basement membrane-like material layered over "pads" of smooth muscle cells. Compared to controls, doxazosin and cholestyramine uniformly reduced the number of foam cells/mm2, foam cell size and ORO staining on average by 66%, 29% and 56%, respectively. We conclude that doxazosin decreases plasma lipids and inhibits the development of the fatty streak to a similar level as cholestyramine treatment.