Jeannie Mui

HO‐1‐mediated macroautophagy: a mechanism for unregulated iron deposition in aging and degenerating neural tissues

J. Neurochem. (2009) 109, 776–791.

Mitochondria-Associated Membrane Formation in Hormone-Stimulated Leydig Cell Steroidogenesis: Role of ATAD3

Leydig cell steroidogenesis is a multistep process that takes place in the mitochondria and endoplasmic reticulum (ER). The physical association between these 2 organelles could facilitate both steroidogenesis substrate availability and mitochondrial product passage to steroidogenic enzymes in the ER, thus regulating the rate of steroid formation. Confocal microscopy, using antisera against organelle-specific antigens, and electron microscopy studies demonstrated that there is an increase in the number of mitochondria-ER contact sites in response to hormone treatment in MA-10 mouse tumor Leydig cells. Electron tomography and 3-dimensional reconstruction allowed for the visualization of mitochondria-associated membranes (MAMs). MAMs were isolated and found to contain the 67-kDa long isoform of the adenosine triphosphatase (ATPase) family, AAA domain-containing protein 3 (ATAD3). The 67-kDa ATAD3 is anchored in the inner mitochondrial membrane and is enriched in outer-inner mitochondrial membrane contact sites. ATAD3-depleted MA-10 cells showed reduced production of steroids in response to human choriogonadotropin but not to 22R-hydroxycholesterol treatment, indicating a role of ATAD3 in the delivery of the substrate cholesterol into the mitochondria. The N terminus of ATAD3 contains 50 amino acids that have been proposed to insert into the outer mitochondrial membrane and associated organelles such as the ER. Deletion of the ATAD3 N terminus resulted in the reduction of hormone-stimulated progesterone biosynthesis, suggesting a role of ATAD3 in mitochondria-ER contact site formation. Taken together, these results demonstrate that the hormone-induced, ATAD3-mediated, MAM formation participates in the optimal transfer of cholesterol from the ER into mitochondria for steroidogenesis.

Schizophrenia-Like Features in Transgenic Mice Overexpressing Human HO-1 in the Astrocytic Compartment

Delineation of key molecules that act epigenetically to transduce diverse stressors into established patterns of disease would facilitate the advent of preventive and disease-modifying therapeutics for a host of neurological disorders. Herein, we demonstrate that selective overexpression of the stress protein heme oxygenase-1 (HO-1) in astrocytes of novel GFAP.HMOX1 transgenic mice results in subcortical oxidative stress and mitochondrial damage/autophagy; diminished neuronal reelin content (males); induction of Nurr1 and Pitx3 with attendant suppression of their targeting miRNAs, 145 and 133b; increased tyrosine hydroxylase and α-synuclein expression with downregulation of the targeting miR-7b of the latter; augmented dopamine and serotonin levels in basal ganglia; reduced D1 receptor binding in nucleus accumbens; axodendritic pathology and altered hippocampal cytoarchitectonics; impaired neurovascular coupling; attenuated prepulse inhibition (males); and hyperkinetic behavior. The GFAP.HMOX1 neurophenotype bears resemblances to human schizophrenia and other neurodevelopmental conditions and implicates glial HO-1 as a prime transducer of inimical (endogenous and environmental) influences on the development of monoaminergic circuitry. Containment of the glial HO-1 response to noxious stimuli at strategic points of the life cycle may afford novel opportunities for the effective management of human neurodevelopmental and neurodegenerative conditions.

Cyanobacterial diversity and activity in modern conical microbialites

Modern conical microbialites are similar to some ancient conical stromatolites, but growth, behavior and diversity of cyanobacteria in modern conical microbialites remain poorly characterized. Here, we analyze the diversity of cyanobacterial 16S rRNA gene sequences in conical microbialites from 14 ponds fed by four thermal sources in Yellowstone National Park and compare cyanobacterial activity in the tips of cones and in the surrounding topographic lows (mats), respectively, by high-resolution mapping of labeled carbon. Cones and adjacent mats contain similar 16S rRNA gene sequences from genetically distinct clusters of filamentous, non-heterocystous cyanobacteria from Subsection III and unicellular cyanobacteria from Subsection I. These sequences vary among different ponds and between two sampling years, suggesting that coniform mats through time and space contain a number of cyanobacteria capable of vertical aggregation, filamentous cyanobacteria incapable of initiating cone formation and unicellular cyanobacteria. Unicellular cyanobacteria are more diverse in topographic lows, where some of these organisms respond to nutrient pulses more rapidly than thin filamentous cyanobacteria. The densest active cyanobacteria are found below the upper 50 μm of the cone tip, whereas cyanobacterial cells in mats are less dense, and are more commonly degraded or encrusted by silica. These spatial differences in cellular activity and density within macroscopic coniform mats imply a strong role for diffusion limitation in the development and the persistence of the conical shape. Similar mechanisms may have controlled the growth, morphology and persistence of small coniform stromatolites in shallow, quiet environments throughout geologic history.

A high-fat diet modulates iron metabolism but does not promote liver fibrosis in hemochromatotic Hjv−/− mice

Hemojuvelin (Hjv) is a membrane protein that controls body iron metabolism by enhancing signaling to hepcidin. Hjv mutations cause juvenile hemochromatosis, a disease of systemic iron overload. Excessive iron accumulation in the liver progressively leads to inflammation and disease, such as fibrosis, cirrhosis, or hepatocellular cancer. Fatty liver (steatosis) may also progress to inflammation (steatohepatitis) and liver disease, and iron is considered as pathogenic cofactor. The aim of this study was to investigate the pathological implications of parenchymal iron overload due to Hjv ablation in the fatty liver. Wild-type (WT) and Hjv(-/-) mice on C57BL/6 background were fed a standard chow, a high-fat diet (HFD), or a HFD supplemented with 2% carbonyl iron (HFD+Fe) for 12 wk. The animals were analyzed for iron and lipid metabolism. As expected, all Hjv(-/-) mice manifested higher serum and hepatic iron and diminished hepcidin levels compared with WT controls. The HFD reduced iron indexes and promoted liver steatosis in both WT and Hjv(-/-) mice. Notably, steatosis was attenuated in Hjv(-/-) mice on the HFD+Fe regimen. Hjv(-/-) animals gained less body weight and exhibited reduced serum glucose and cholesterol levels. Histological and ultrastructural analysis revealed absence of iron-induced inflammation or liver fibrosis despite early signs of liver injury (expression of α-smooth muscle actin). We conclude that parenchymal hepatic iron overload does not suffice to trigger progression of liver steatosis to steatohepatitis or fibrosis in C57BL/6 mice.

Reaction-diffusion model of nutrient uptake in a biofilm: theory and experiment.

Microbes in natural settings typically live attached to surfaces in complex communities called biofilms. Despite the many advantages of biofilm formation, communal living forces microbes to compete with one another for resources. Here we combine mathematical models with stable isotope techniques to test a reaction-diffusion model of competition in a photosynthetic biofilm. In this model, a nutrient is transported through the mat by diffusion and is consumed at a rate proportional to its local concentration. When the nutrient is supplied from the surface of the biofilm, the balance between diffusion and consumption gives rise to gradients of nutrient availability, resulting in gradients of nutrient uptake. To test this model, a biofilm was incubated for a fixed amount of time with an isotopically labeled nutrient that was incorporated into cellular biomass. Thus, the concentration of labeled nutrient in a cell is a measure of the mean rate of nutrient incorporation over the course of the experiment. Comparison of this measurement to the solution of the reaction-diffusion model in the biofilm confirms the presence of gradients in nutrient uptake with the predicted shape. The excellent agreement between theory and experiment lends strong support to this one-parameter model of reaction and diffusion of nutrients in a biofilm. Having validated this model empirically, we discuss how these dynamics may arise from diffusion through a reactive heterogeneous medium. More generally, this result identifies stable isotope techniques as a powerful tool to test quantitative models of chemical transport through biofilms.

Ultrastructural Characterization of Turnip Mosaic Virus-Induced Cellular Rearrangements Reveals Membrane-Bound Viral Particles Accumulating in Vacuoles

ABSTRACT Positive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K2 plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2 tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation. IMPORTANCE Positive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The ultrastructure of turnip mosaic virus (TuMV)-induced membrane remodeling was investigated over several days of infection. The first change that was observed involved endoplasmic reticulum-connected convoluted membrane accumulation. This was followed by the formation of single-membrane tubules, which were shown to be viral RNA replication sites. Later in the infection process, double-membrane tubular structures were observed and were associated with viral particle bundles. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. This work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.

Choroideremia Is a Systemic Disease With Lymphocyte Crystals and Plasma Lipid and RBC Membrane Abnormalities

PURPOSE Photoreceptor neuronal degenerations are common, incurable causes of human blindness affecting 1 in 2000 patients worldwide. Only half of all patients are associated with known mutations in over 250 disease genes, prompting our research program to identify the remaining new genes. Most retinal degenerations are restricted to the retina, but photoreceptor degenerations can also be found in a wide variety of systemic diseases. We identified an X-linked family from Sri Lanka with a severe choroidal degeneration and postulated a new disease entity. Because of phenotypic overlaps with Biettis crystalline dystrophy, which was recently found to have systemic features, we hypothesized that a systemic disease may be present in this new disease as well. METHODS For phenotyping, we performed detailed eye exams with in vivo retinal imaging by optical coherence tomography. For genotyping, we performed whole exome sequencing, followed by Sanger sequencing confirmations and cosegregation. Systemic investigations included electron microscopy studies of peripheral blood cells in patients and in normal controls and detailed fatty acid profiles (both plasma and red blood cell [RBC] membranes). Fatty acid levels were compared to normal controls, and only values two standard deviations above or below normal controls were further evaluated. RESULTS The family segregated a REP1 mutation, suggesting choroideremia (CHM). We then found crystals in peripheral blood lymphocytes and discovered significant plasma fatty acid abnormalities and RBC membrane abnormalities (i.e., elevated plasmalogens). To replicate our discoveries, we expanded the cohort to nine CHM patients, genotyped them for REP1 mutations, and found the same abnormalities (crystals and fatty acid abnormalities) in all patients. CONCLUSIONS Previously, CHM was thought to be restricted to the retina. We show, to our knowledge for the first time, that CHM is a systemic condition with prominent crystals in lymphocytes and significant fatty acid abnormalities.

Visualization of SNARE-Mediated Organelle Membrane Hemifusion by Electron Microscopy

SNARE-mediated membrane fusion is required for membrane trafficking as well as organelle biogenesis and homeostasis. The membrane fusion reaction involves sequential formation of hemifusion intermediates, whereby lipid monolayers partially mix on route to complete bilayer merger. Studies of the Saccharomyces cerevisiae lysosomal vacuole have revealed many of the fundamental mechanisms that drive the membrane fusion process, as well as features unique to organelle fusion. However, until recently, it has not been amenable to electron microscopy methods that have been invaluable for studying hemifusion in other model systems. Herein, we describe a method to visualize hemifusion intermediates during homotypic vacuole membrane fusion in vitro by transmission electron microscopy (TEM), electron tomography, and cryogenic electron microscopy (cryoEM). This method facilitates acquisition of invaluable ultrastructural data needed to comprehensively understand how fusogenic lipids and proteins contribute to SNARE-mediated membrane fusion-by-hemifusion and the unique features of organelle versus small-vesicle fusion.

Mouse models of hereditary hemochromatosis do not develop early liver fibrosis in response to a high fat diet

Hepatic iron overload, a hallmark of hereditary hemochromatosis (HH), triggers progressive liver disease. There is also increasing evidence for a pathogenic role of iron in non-alcoholic fatty liver disease (NAFLD), which may progress to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular cancer. Mouse models of HH and NAFLD can be used to explore potential interactions between iron and lipid metabolic pathways. Hfe−/− mice, a model of moderate iron overload, were reported to develop early liver fibrosis in response to a high fat diet. However, this was not the case with Hjv−/− mice, a model of severe iron overload. These data raised the possibility that the Hfe gene may protect against liver injury independently of its iron regulatory function. Herein, we addressed this hypothesis in a comparative study utilizing wild type, Hfe−/−, Hjv−/− and double Hfe−/−Hjv−/− mice. The animals, all in C57/BL6 background, were fed with a high fat diet for 14 weeks and developed hepatic steatosis, associated with mild iron overload. Hfe co-ablation did not sensitize steatotic Hjv-deficient mice to liver injury. Moreover, we did not observe any signs of liver inflammation or fibrosis even in single steatotic Hfe−/− mice. Ultrastructural studies revealed a reduced lipid and glycogen content in Hjv−/− hepatocytes, indicative of a metabolic defect. Interestingly, glycogen levels were restored in double Hfe−/−Hjv−/− mice, which is consistent with a metabolic function of Hfe. We conclude that hepatocellular iron excess does not aggravate diet-induced steatosis to steatohepatitis or early liver fibrosis in mouse models of HH, irrespectively of the presence or lack of Hfe.