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.

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.

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.

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.

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.

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.

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.

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.

Synthesis and characterization of arylamine derivatives of rauwolscine as molecular probes for alpha 2-adrenergic receptors.

The selective alpha 2-adrenergic receptor antagonist rauwolscine was structurally modified to yield a series of arylamine carboxamide derivatives, which were investigated as potential molecular probes for the localization and structural characterization of alpha 2-adrenergic receptors. The arylamine carboxamides differ in the number of carbon atoms separating the reactive phenyl moiety from the fused ring structure of the parent compound, rauwolscine carboxylate. Competitive inhibition studies with [3H]rauwolscine in rat kidney membranes indicate that the affinity for the carboxamide derivatives is inversely related to the length of the carbon spacer arm with rauwolscine 4-aminophenyl carboxamide (zero carbon spacer arm; rau-AMPC) exhibiting the highest affinity (Kd = 2.3 +/- 0.2 nM). Radioiodination of rau-AMPC yields a ligand, 125I-rau-AMPC, which binds to rat kidney alpha 2-adrenergic receptors with high affinity, as determined by both kinetic analysis (Kd = k2/k1 = 0.016 min-1/2.1 X 10(7) M-1 min-1 = 0.76 nM) and equilibrium binding studies (Kd = 0.78 +/- 0.16 nM). 125I-rau-AMPC was quantitatively converted to the photolabile arylazide derivative 17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-azido-3-[125I]iodophenyl) carboxamide (125I-rau-AZPC). In a partially purified receptor preparation from porcine brain, this compound photolabels a major (Mr = 62,000) peptide. The labeling of this peptide is inhibited by adrenergic agonists and antagonists with a rank order of potency consistent with an alpha 2-adrenergic receptor binding site. Both 125I-rau-AMPC and the photolabile arylazide derivative, 125I-rau-AZPC, should prove useful as molecular probes for the structural and biochemical characterization of alpha 2-adrenergic receptors.

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.

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.

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.

Quantitation of oil red O staining of the aorta in hypercholesterolemic rats.

A technique is described which provides morphologic and quantitative data on the amount of oil red O (ORO) staining in thoracic aortas of rats fed a high cholesterol diet. Samples are stained with ORO, the dye is extracted, and the concentration of ORO in the extract is measured colorimetrically. Wistar rats fed ad libitum either standard chow (control group: n = 15) or chow supplemented with 4% cholesterol, 1% cholic acid, and 0.5% thiouracil (CCT group: n = 23) were maintained on these diets for 1, 3, 6, 9, or 12 months. Plasma cholesterol levels averaged overall 87 and 737 mg/dl for the control and CCT groups, respectively. Animals were killed under anesthesia by perfusion fixation with formalin or glutaraldehyde, and samples of thoracic aorta were stained with ORO. After microscopic study en face and measurement of surface area, the ORO was extracted in chloroform-methanol (2:1). Concentrations of ORO (microM) were determined from a standard curve and expressed as microM/mm2 of aorta. Aortas of CCT animals showed progressive diet- and time-dependent increases in the amount of ORO staining compared to controls. We conclude that this method yields reliable quantitative data applicable to studying atherosclerosis in small animals.

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.

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.

Purification of the alpha 2-adrenergic receptor from porcine brain using a yohimbine-agarose affinity matrix.

A procedure has been developed for purification of the porcine brain alpha 2-adrenergic receptor to homogeneity. alpha 2-Adrenergic receptors were solubilized from porcine brain particulate preparations using sequential extraction into sodium cholate- and digitonin-containing buffers. The alpha 2-adrenergic receptors in the digitonin extract were identified using the alpha 2-adrenergic selective antagonist, [3H]yohimbine, and demonstrated the same specificity for interaction with adrenergic ligands as did the receptors in particulate preparations. Extraction into digitonin-containing buffers eliminated the modulation of receptor-agonist interactions by guanine nucleotides, but not by monovalent cations. A novel affinity resin, yohimbine-agarose, was synthesized and used for purification of alpha 2-adrenergic receptors. Using two sequential yohimbine-agarose affinity chromatography steps, digitonin-solubilized alpha 2-adrenergic receptors from porcine brain cortex were purified to homogeneity as assessed by radioiodination and silver stain analysis of these preparations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified alpha 2-adrenergic receptor has an approximate Mr = 65,000, as determined by photolabeling of the adrenergic ligand-binding subunit. The yohimbine-agarose affinity resin should be useful for purifying quantities of receptor sufficient for studies of receptor structure and function.

Determination of the state and content of water in human normal and cataractous lenses by differential scanning calorimetry.

The state of water in normal and cataractous human lenses was determined by differential scanning calorimetry. Results indicate that water 'bound' to the lens proteins decreases during the process of cataract formation. These findings are consistent with the documented knowledge concerning cataract-related changes in the lens proteins.

Determination of the state and content of water in normal avian, fish, porcine, bovine, and human lenses as studied by differential scanning calorimetry.

Differential scanning calorimetry was used to measure the relative amounts of 'bulk' and 'bound' water in normal avian, bovine, fish, human, and porcine lenses. The amounts of bound water (mg bound water/mg lens dry weight) found in avian and porcine lenses were statistically different from each other in addition to being statistically different from fish, human, and bovine lenses. There were no significant differences in the mean values between human, fish, and bovine lenses. Avian lenses had the highest amount of bound water, while fish lenses had the lowest bound water content. Significant differences in total water content (mg total water/mg lens dry weight) were observed between all of the lenses, with the exception of bovine and human lenses which were not statistically different. Fish lenses had the lowest amount of total water, and avian lenses had the highest total water content. There were significant differences in bulk water content (mg bulk water/mg lens dry weight) between all of the lenses. Avian lenses had the highest bulk water content, and fish lenses had the lowest bulk water content.

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.