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, 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 hereditary hemochromatosis 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 C57BL/6J background, were fed with high fat diets for 14 weeks and developed hepatic steatosis, associated with 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 hereditary hemochromatosis, irrespectively of the presence or lack of Hfe.

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.

Transferrin receptor 1 controls systemic iron homeostasis by fine-tuning hepcidin expression to hepatocellular iron load.

Transferrin receptor 1 (Tfr1) mediates uptake of circulating transferrin-bound iron to developing erythroid cells and other cell types. Its critical physiological function is highlighted by the embryonic lethal phenotype of Tfr1-knockout (Tfrc-/-) mice and the pathologies of several tissue-specific knockouts. We generated TfrcAlb-Cre mice bearing hepatocyte-specific ablation of Tfr1 to explore implications in hepatocellular and systemic iron homeostasis. TfrcAlb-Cre mice are viable and do not display any apparent liver pathology. Nevertheless, their liver iron content (LIC) is lower compared with that of control Tfrcfl/fl littermates as a result of the reduced capacity of Tfr1-deficient hepatocytes to internalize iron from transferrin. Even though liver Hamp messenger RNA (mRNA) and serum hepcidin levels do not differ between TfrcAlb-Cre and Tfrcfl/fl mice, Hamp/LIC and hepcidin/LIC ratios are significantly higher in the former. Importantly, this is accompanied by modest hypoferremia and microcytosis, and it predisposes TfrcAlb-Cre mice to iron-deficiency anemia. TfrcAlb-Cre mice appropriately regulate Hamp expression following dietary iron manipulations or holo-transferrin injection. Holo-transferrin also triggers proper induction of Hamp mRNA, ferritin, and Tfr2 in primary TfrcAlb-Cre hepatocytes. We further show that these cells can acquire 59Fe from 59Fe-transferrin, presumably via Tfr2. We conclude that Tfr1 is redundant for basal hepatocellular iron supply but essential for fine-tuning hepcidin responses according to the iron load of hepatocytes. Our data are consistent with an inhibitory function of Tfr1 on iron signaling to hepcidin via its interaction with Hfe. Moreover, they highlight hepatocellular Tfr1 as a link between cellular and systemic iron-regulatory pathways.

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

UNLABELLED: 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 Bietti's 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.

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.

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.

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.

Unregulated brain iron deposition in transgenic mice over-expressing HMOX1 in the astrocytic compartment.

The mechanisms responsible for pathological iron deposition in the aging and degenerating mammalian CNS remain poorly understood. The stress protein, HO-1 mediates the degradation of cellular heme to biliverdin/bilirubin, free iron, and CO and is up-regulated in the brains of persons with Alzheimer's disease and Parkinson's disease. HO-1 induction in primary astroglial cultures promotes deposition of non-transferrin iron, mitochondrial damage and macroautophagy, and predisposes cocultured neuronal elements to oxidative injury. To gain a better appreciation of the role of glial HO-1 in vivo, we probed for aberrant brain iron deposition using Perls' method and dynamic secondary ion mass spectrometry in novel, conditional GFAP.HMOX1 transgenic mice that selectively over-express human HO-1 in the astrocytic compartment. At 48 weeks, the GFAP.HMOX1 mice exhibited increased deposits of glial iron in hippocampus and other subcortical regions without overt changes in iron-regulatory and iron-binding proteins relative to age-matched wild-type animals. Dynamic secondary ion mass spectrometry revealed abundant FeO⁻ signals in the transgenic, but not wild-type, mouse brain that colocalized to degenerate mitochondria and osmiophilic cytoplasmic inclusions (macroautophagy) documented by TEM. Sustained up-regulation of HO-1 in astrocytes promotes pathological brain iron deposition and oxidative mitochondrial damage characteristic of Alzheimer's disease-affected neural tissues. Curtailment of glial HO-1 hyperactivity may limit iron-mediated cytotoxicity in aging and degenerating neural tissues.

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.

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

Oxidative stress, deposition of non-transferrin iron, and mitochondrial insufficiency occur in the brains of patients with Alzheimer disease (AD) and Parkinson disease (PD). We previously demonstrated that heme oxygenase-1 (HO-1) is up-regulated in AD and PD brain and promotes the accumulation of non-transferrin iron in astroglial mitochondria. Herein, dynamic secondary ion mass spectrometry (SIMS) and other techniques were employed to ascertain (i) the impact of HO-1 over-expression on astroglial mitochondrial morphology in vitro, (ii) the topography of aberrant iron sequestration in astrocytes over-expressing HO-1, and (iii) the role of iron regulatory proteins (IRP) in HO-1-mediated iron deposition. Astroglial hHO-1 over-expression induced cytoplasmic vacuolation, mitochondrial membrane damage, and macroautophagy. HO-1 promoted trapping of redox-active iron and sulfur within many cytopathological profiles without impacting ferroportin, transferrin receptor, ferritin, and IRP2 protein levels or IRP1 activity. Thus, HO-1 activity promotes mitochondrial macroautophagy and sequestration of redox-active iron in astroglia independently of classical iron mobilization pathways. Glial HO-1 may be a rational therapeutic target in AD, PD, and other human CNS conditions characterized by the unregulated deposition of brain iron.

Maintenance of axo-oligodendroglial paranodal junctions requires DCC and netrin-1.

Paranodal axoglial junctions are essential for the segregation of myelinated axons into distinct domains and efficient conduction of action potentials. Here, we show that netrin-1 and deleted in colorectal cancer (DCC) are enriched at the paranode in CNS myelin. We then address whether netrin-1 signaling influences paranodal adhesion between oligodendrocytes and axons. In the absence of netrin-1 or DCC function, oligodendroglial paranodes initially develop and mature normally but later become disorganized. Lack of DCC or netrin-1 resulted in detachment of paranodal loops from the axonal surface and the disappearance of transverse bands. Furthermore, the domain organization of myelin is compromised in the absence of netrin-1 signaling: K+ channels inappropriately invade the paranodal region, and the normally restricted paranodal distribution of Caspr expands longitudinally along the axon. Our findings identify an essential role for netrin-1 and DCC regulating the maintenance of axoglial junctions.

Gold-labeled block copolymer micelles reveal gold aggregates at multiple subcellular sites.

There is increasing interest in the usefulness of block copolymer micelles as drug delivery vehicles. However, their subcellular distribution has not been explored extensively, mostly because of the lack of adequately labeled block copolymers. In a previous study, we showed that fluorescently labeled block copolymer micelles entered living cells and co-localized with cytoplasmic organelles selectively labeled with fluorescent dyes. The details of the observed co-localizations were, however, limited by the resolution of the fluorescence approach, which is ca. 500 nm. Using transmission electron microscopy (TEM), we established time- and concentration-dependent subcellular distributions of gold-labeled micelles within human embryonic kidney (HEK 293) cells and human lung carcinoma (A549) cells. Gold particles were incorporated into poly(4-vinylpyridine)-block-poly(ethylene oxide) (P4VP21-b-PEO45) micelles. Data from dynamic light scattering (DLS) and TEM analyses revealed that the sizes of the gold particles ranged from 4 to 8 nm. The cells survived up to 24 h in the presence of low gold-labeled micelle concentrations (0.73 microg/mL), but cell death occurred at higher concentrations (i.e., kidney cells are more susceptible than lung cells). Over 24 h periods of equivalent exposure, lung cells internalized significantly more gold-incorporated micelles than kidney cells. Although micelles were added to the cell culture media as dispersed colloidal particles, the presence of serum in these media caused aggregation. These aggregates occurred mainly close to the cell plasma membrane at early times (5-10 min); however, at later times (24 h) aggregated particles were seen inside endosomes and lysozomes. Thus, gold-incorporated (labeled) micelles can serve as a valuable extension of the fluorescence approach to visualizing the localization of micelles in subcellular compartments, improving the resolution by at least 20-fold.

Characterization of cell- and region-specific abnormalities in the epididymis of cathepsin A deficient mice.

Cathepsin A (PPCA), a lysosomal carboxypeptidase that functions as a protective protein for alpha-neuraminidase and beta-galactosidase in a multi-enzyme complex, has been shown to be expressed in the epithelial cells of the epididymis. In the present study, the epididymis of PPCA-/- mice from 2 to 10 months of age was compared with those of their wild-type counterparts. Major accumulations of pale vacuoles, corresponding to lysosomes, were noted in principal and narrow/apical cells in PPCA-/- mice, and clear cells also appearing highly vacuolated, were grossly enlarged in size. This was especially evident in the caput and corpus regions, where quantitative analyses confirmed that the epithelium of the tubules in these regions was expanding in profile area. In addition, the base of the epithelium in these regions was often greatly vacuolated, corresponding to cells that presented no identifiable features and appeared to be degenerating. Halo cells dispersed at various levels in the epithelium also appeared to be abnormal, accumulating pale lysosomes. Furthermore, numerous macrophages were observed in the intertubular space of the entire duct, presenting a large size and plethora of pale lysosomes. Taken together, the present data indicate major lysosomal abnormalities in the epididymis of PPCA-/- mice in a cell type and region specific manner. In addition, it is suggested that the compromised halo cells, due to PPCA deficiency within their lysosomes, cannot function properly and as a result there is a recruitment of macrophages in the intertubular space.

Hfe deficiency increases susceptibility to cardiotoxicity and exacerbates changes in iron metabolism induced by doxorubicin.

The clinical use of doxorubicin (DOX), an anthracycline chemotherapeutic agent, is limited by cardiotoxicity. The possible involvement of iron in DOX-induced cardiotoxicity became evident from studies in which iron chelators were shown to be cardioprotective. Iron overload is found in hereditary hemochromatosis, a genetic disorder prevalent in individuals of European descent. We hypothesized that Hfe deficiency may increase susceptibility to DOX-induced toxicity. Acute cardiotoxicity and iron changes were studied after treatment with DOX in Hfe knock-out (Hfe-/-) mice and wild-type mice. DOX-induced iron metabolism changes were intensified in Hfe-/- mice, which accumulated significantly more iron in the heart, liver, and pancreas, but less in the spleen compared with wild-type mice. In addition, Hfe-deficient mice exhibited significantly greater sensitivity to DOX-induced elevations in serum creatine kinase and aspartate aminotransferase. Increased mortality after chronic DOX treatment was observed in Hfe-/- mice and Hfe+/-mice compared with wild-type mice. DOX-treated Hfe-/- mice had a higher degree of mitochondrial damage and iron deposits in the heart than did wild-type mice. These data demonstrate that Hfe deficiency in mice increases susceptibility to DOX-induced cardiotoxicity and suggest that genetic mutations related to defects in iron metabolism may contribute to its cardiotoxicity in humans.

Osteopontin expression and regulation in the testis, efferent ducts, and epididymis of rats during postnatal development through to adulthood.

Osteopontin (OPN), a multifunctional phosphoprotein found in both hard and soft tissues, was examined in the male reproductive tract. The expression and regulation of OPN in the rat testis, efferent ducts, and epididymis was examined during postnatal development through to adulthood using immunocytochemistry at the light- and electron-microscopic level. Immunoblot analysis revealed a major 30-kDa band for epididymal tissue and a major 60-kDa band for the testis. In the testis, immunostaining of OPN was noted in early germ cells from spermatogonia to early pachytene spermatocytes, suggesting a role for OPN as an adhesive protein binding these cells to the basement membrane and adjacent Sertoli cells. Nonciliated cells of the efferent ducts expressed OPN, whereas a cell- and region-specific distribution of OPN was observed in the epididymis. Reactivity of OPN in the apical region of the cell corresponded to labeling of microvilli, small endocytic vesicles, and endosomes, where OPN may serve to remove calcium from the epididymal lumen and, thus, prevent mineral accumulation and subsequent decrease in sperm fertility. Regulation and postnatal studies revealed that circulating androgens regulate OPN expression in principal cells of the epididymis only. Taken together, the data reveal cell- and region-specific expression and regulation of OPN in the epididymis.