Large analysis of genetic manipulations reveals an inverse correlation between initial alcohol resistance and rapid tolerance phenotypes.

Tolerance occurs when, following an initial experience with a substance, more of the substance is required subsequently to induce identical behavioral effects. Tolerance is not well-understood, and numerous researchers have turned to model organisms, particularly Drosophila melanogaster, to unravel its mechanisms. Flies have high translational relevance for human alcohol responses, and there is substantial overlap in disease-causing genes between flies and humans, including those associated with Alcohol Use Disorder. Numerous Drosophila tolerance mutants have been described; however, approaches used to identify and characterize these mutants have varied across time and labs and have mostly disregarded any impact of initial resistance/sensitivity to ethanol on subsequent tolerance development. Here, we analyzed our own, as well as data published by other labs to uncover an inverse correlation between initial ethanol resistance and tolerance phenotypes. This inverse correlation suggests that initial resistance phenotypes can explain many 'perceived' tolerance phenotypes, thus classifying such mutants as 'secondary' tolerance mutants. Additionally, we show that tolerance should be measured as a relative increase in time to sedation between an initial and second exposure rather than an absolute change in time to sedation. Finally, based on our analysis, we provide a method for using a linear regression equation to assess the residuals of potential tolerance mutants. These residuals provide predictive insight into the likelihood of a mutant being a 'primary' tolerance mutant, where a tolerance phenotype is not solely a consequence of initial resistance, and we offer a framework for understanding the relationship between initial resistance and tolerance.

Synaptic Mechanisms of Ethanol Tolerance and Neuroplasticity: Insights from Invertebrate Models.

Alcohol tolerance is a neuroadaptive response that leads to a reduction in the effects of alcohol caused by previous exposure. Tolerance plays a critical role in the development of alcohol use disorder (AUD) because it leads to the escalation of drinking and dependence. Understanding the molecular mechanisms underlying alcohol tolerance is therefore important for the development of effective therapeutics and for understanding addiction in general. This review explores the molecular basis of alcohol tolerance in invertebrate models, Drosophila and C. elegans, focusing on synaptic transmission. Both organisms exhibit biphasic responses to ethanol and develop tolerance similar to that of mammals. Furthermore, the availability of several genetic tools makes them a great candidate to study the molecular basis of ethanol response. Studies in invertebrate models show that tolerance involves conserved changes in the neurotransmitter systems, ion channels, and synaptic proteins. These neuroadaptive changes lead to a change in neuronal excitability, most likely to compensate for the enhanced inhibition by ethanol.

Low-Iron Diet-Induced Fatty Liver Development Is Microbiota Dependent and Exacerbated by Loss of the Mitochondrial Iron Importer Mitoferrin2.

Iron deficiency is the number one nutritional problem worldwide. Iron uptake is regulated at the intestine and is highly influenced by the gut microbiome. Blood from the intestines drains directly into the liver, informing iron status and gut microbiota status. Changes in either iron or the microbiome are tightly correlated with the development of metabolic dysfunction-associated steatotic liver disease (MASLD). To investigate the underlying mechanisms of the development of MASLD that connect altered iron metabolism and gut microbiota, we compared specific pathogen free (SPF) or germ-free (GF) mice, fed a normal or low-iron diet. SPF mice on a low-iron diet showed reduced serum triglycerides and MASLD. In contrast, GF low-iron diet-fed mice showed increased serum triglycerides and did not develop hepatic steatosis. SPF mice showed significant changes in liver lipid metabolism and increased insulin resistance that was dependent upon the presence of the gut microbiota. We report that total body loss of mitochondrial iron importer Mitoferrin2 (Mfrn2-/-) exacerbated the development of MASLD on a low-iron diet with significant lipid metabolism alterations. Our study demonstrates a clear contribution of the gut microbiome, dietary iron, and Mfrn2 in the development of MASLD and metabolic syndrome.

Large genetic analysis of alcohol resistance and tolerance reveals an inverse correlation and suggests 'true' tolerance mutants.

Tolerance occurs when, following an initial experience with a substance, more of the substance is required subsequently to induce the same behavioral effects. Tolerance is historically not well-understood, and numerous researchers have turned to model organisms, particularly Drosophila melanogaster, to unravel its mechanisms. Flies have high translational relevance for human alcohol responses, and there is substantial overlap in disease-causing genes between flies and humans, including those associated with Alcohol Use Disorder. Numerous Drosophila tolerance mutants have been described; however, approaches used to identify and characterize these mutants have varied across time and between labs and have mostly disregarded any impact of initial resistance/sensitivity to ethanol on subsequent tolerance development. Here, we have analyzed a large amount of data - our own published and unpublished data and data published by other labs - to uncover an inverse correlation between initial ethanol resistance and tolerance phenotypes. This inverse correlation suggests that initial resistance phenotypes can explain many 'perceived' tolerance phenotypes. Additionally, we show that tolerance should be measured as a relative increase in time to sedation between an initial and second exposure rather than an absolute change in time to sedation. Finally, based on our analysis, we provide a method for using a linear regression equation to assess the residuals of potential tolerance mutants. We show that these residuals provide predictive insight into the likelihood of a mutant being a 'true' tolerance mutant, and we offer a framework for understanding the relationship between initial resistance and tolerance.

Loss of the mitochondrial protein Abcb10 results in altered arginine metabolism in MEL and K562 cells and nutrient stress signaling through ATF4.

Abcb10 is a mitochondrial membrane protein involved in hemoglobinization of red cells. Abcb10 topology and ATPase domain localization suggest it exports a substrate, likely biliverdin, out of mitochondria that is necessary for hemoglobinization. In this study, we generated Abcb10 deletion cell lines in both mouse murine erythroleukemia and human erythroid precursor human myelogenous leukemia (K562) cells to better understand the consequences of Abcb10 loss. Loss of Abcb10 resulted in an inability to hemoglobinize upon differentiation in both K562 and mouse murine erythroleukemia cells with reduced heme and intermediate porphyrins and decreased levels of aminolevulinic acid synthase 2 activity. Metabolomic and transcriptional analyses revealed that Abcb10 loss gave rise to decreased cellular arginine levels, increased transcripts for cationic and neutral amino acid transporters with reduced levels of the citrulline to arginine converting enzymes argininosuccinate synthetase and argininosuccinate lyase. The reduced arginine levels in Abcb10-null cells gave rise to decreased proliferative capacity. Arginine supplementation improved both Abcb10-null proliferation and hemoglobinization upon differentiation. Abcb10-null cells showed increased phosphorylation of eukaryotic translation initiation factor 2 subunit alpha, increased expression of nutrient sensing transcription factor ATF4 and downstream targets DNA damage inducible transcript 3 (Chop), ChaC glutathione specific gamma-glutamylcyclotransferase 1 (Chac1), and arginyl-tRNA synthetase 1 (Rars). These results suggest that when the Abcb10 substrate is trapped in the mitochondria, the nutrient sensing machinery is turned on remodeling transcription to block protein synthesis necessary for proliferation and hemoglobin biosynthesis in erythroid models.

The mitochondrial metal transporters mitoferrin1 and mitoferrin2 are required for liver regeneration and cell proliferation in mice.

Mitochondrial iron import is essential for iron-sulfur cluster formation and heme biosynthesis. Two nuclear-encoded vertebrate mitochondrial high-affinity iron importers, mitoferrin1 (Mfrn1) and Mfrn2, have been identified in mammals. In mice, the gene encoding Mfrn1, solute carrier family 25 member 37 (Slc25a37), is highly expressed in sites of erythropoiesis, and whole-body Slc25a37 deletion leads to lethality. Here, we report that mice with a deletion of Slc25a28 (encoding Mfrn2) are born at expected Mendelian ratios, but show decreased male fertility due to reduced sperm numbers and sperm motility. Mfrn2-/- mice placed on a low-iron diet exhibited reduced mitochondrial manganese, cobalt, and zinc levels, but not reduced iron. Hepatocyte-specific loss of Slc25a37 (encoding Mfrn1) in Mfrn2-/- mice did not affect animal viability, but resulted in a 40% reduction in mitochondrial iron and reduced levels of oxidative phosphorylation proteins. Placing animals on a low-iron diet exaggerated the reduction in mitochondrial iron observed in liver-specific Mfrn1/2-knockout animals. Mfrn1-/-/Mfrn2-/- bone marrow-derived macrophages or skin fibroblasts in vitro were unable to proliferate, and overexpression of Mfrn1-GFP or Mfrn2-GFP prevented this proliferation defect. Loss of both mitoferrins in hepatocytes dramatically reduced regeneration in the adult mouse liver, further supporting the notion that both mitoferrins transport iron and that their absence limits proliferative capacity of mammalian cells. We conclude that Mfrn1 and Mfrn2 contribute to mitochondrial iron homeostasis and are required for high-affinity iron import during active proliferation of mammalian cells.

Altered Actin Filament Dynamics in the Drosophila Mushroom Bodies Lead to Fast Acquisition of Alcohol Consumption Preference.

Alcohol use is highly prevalent in the United States and across the world, and every year millions of people suffer from alcohol use disorders (AUDs). Although the genetic contribution to developing AUDs is estimated to be 50-60%, many of the underlying molecular mechanisms remain unclear. Previous studies from our laboratory revealed that Drosophila melanogaster lacking RhoGAP18B and Ras Suppressor 1 (Rsu1) display reduced sensitivity to ethanol-induced sedation. Both Rsu1 and RhoGAP18B are negative regulators of the small Rho-family GTPase, Rac1, a modulator of actin dynamics. Here we investigate the role of Rac1 and its downstream target, the actin-severing protein cofilin, in alcohol consumption preference. We show that these two regulators of actin dynamics can alter male experience-dependent alcohol preference in a bidirectional manner: expressing either activated Rac1 or dominant-negative cofilin in the mushroom bodies (MBs) abolishes experience-dependent alcohol preference. Conversely, dominant-negative Rac1 or activated cofilin MB expression lead to faster acquisition of alcohol preference. Our data show that Rac1 and cofilin activity are key to determining the rate of acquisition of alcohol preference, revealing a critical role of actin dynamics regulation in the development of voluntary self-administration in DrosophilaSIGNIFICANCE STATEMENT The risks for developing an alcohol use disorder (AUD) are strongly determined by genetic factors. Understanding the genes and molecular mechanisms that contribute to that risk is therefore a necessary first step for the development of targeted therapeutic intervention. Here we show that regulators of actin cytoskeleton dynamics can bidirectionally determine the acquisition rate of alcohol self-administration, highlighting this process as a key mechanism contributing to the risk of AUD development.

Reductions in the mitochondrial ABC transporter Abcb10 affect the transcriptional profile of heme biosynthesis genes.

ATP-binding cassette subfamily B member 10 (Abcb10) is a mitochondrial ATP-binding cassette (ABC) transporter that complexes with mitoferrin1 and ferrochelatase to enhance heme biosynthesis in developing red blood cells. Reductions in Abcb10 levels have been shown to reduce mitoferrin1 protein levels and iron import into mitochondria, resulting in reduced heme biosynthesis. As an ABC transporter, Abcb10 binds and hydrolyzes ATP, but its transported substrate is unknown. Here, we determined that decreases in Abcb10 did not result in protoporphyrin IX accumulation in morphant-treated zebrafish embryos or in differentiated Abcb10-specific shRNA murine Friend erythroleukemia (MEL) cells in which Abcb10 was specifically silenced with shRNA. We also found that the ATPase activity of Abcb10 is necessary for hemoglobinization in MEL cells, suggesting that the substrate transported by Abcb10 is important in mediating increased heme biosynthesis during erythroid development. Inhibition of 5-aminolevulinic acid dehydratase (EC 4.2.1.24) with succinylacetone resulted in both 5-aminolevulinic acid (ALA) accumulation in control and Abcb10-specific shRNA MEL cells, demonstrating that reductions in Abcb10 do not affect ALA export from mitochondria and indicating that Abcb10 does not transport ALA. Abcb10 silencing resulted in an alteration in the heme biosynthesis transcriptional profile due to repression by the transcriptional regulator Bach1, which could be partially rescued by overexpression of Alas2 or Gata1, providing a mechanistic explanation for why Abcb10 shRNA MEL cells exhibit reduced hemoglobinization. In conclusion, our findings rule out that Abcb10 transports ALA and indicate that Abcb10's ATP-hydrolysis activity is critical for hemoglobinization and that the substrate transported by Abcb10 provides a signal that optimizes hemoglobinization.

A Yeast/Drosophila Screen to Identify New Compounds Overcoming Frataxin Deficiency.

Friedreich's ataxia (FA) is a rare neurodegenerative disease which is very debilitating for the patients who progressively lose their autonomy. The lack of efficient therapeutic treatment of the disease strongly argues for urgent need to search for new active compounds that may stop the progression of the disease or prevent the appearance of the symptoms when the genetic defect is diagnosed early enough. In the present study, we used a yeast strain with a deletion of the frataxin homologue gene as a model of FA cells in a primary screen of two chemical libraries, a fraction of the French National Chemical Library (5500 compounds) and the Prestwick collection (880 compounds). We ran a secondary screen on Drosophila melanogaster flies expressing reduced levels of frataxin during larval development. Half of the compounds selected in yeast appeared to be active in flies in this developmental paradigm, and one of the two compounds with highest activities in this assay partially rescued the heart dilatation phenotype resulting from heart specific depletion of frataxin. The unique complementarity of these two frataxin-deficient models, unicellular and multicellular, appears to be very efficient to select new compounds with improved selectivity, bringing significant perspectives towards improvements in FA therapy.

Snx3 regulates recycling of the transferrin receptor and iron assimilation.

Sorting of endocytic ligands and receptors is critical for diverse cellular processes. The physiological significance of endosomal sorting proteins in vertebrates, however, remains largely unknown. Here we report that sorting nexin 3 (Snx3) facilitates the recycling of transferrin receptor (Tfrc) and thus is required for the proper delivery of iron to erythroid progenitors. Snx3 is highly expressed in vertebrate hematopoietic tissues. Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates due to impaired transferrin (Tf)-mediated iron uptake and its accumulation in early endosomes. This impaired iron assimilation can be complemented with non-Tf iron chelates. We show that Snx3 and Vps35, a component of the retromer, interact with Tfrc to sort it to the recycling endosomes. Our findings uncover a role of Snx3 in regulating Tfrc recycling, iron homeostasis, and erythropoiesis. Thus, the identification of Snx3 provides a genetic tool for exploring erythropoiesis and disorders of iron metabolism.

Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts.

Defects in the availability of haem substrates or the catalytic activity of the terminal enzyme in haem biosynthesis, ferrochelatase (Fech), impair haem synthesis and thus cause human congenital anaemias. The interdependent functions of regulators of mitochondrial homeostasis and enzymes responsible for haem synthesis are largely unknown. To investigate this we used zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anaemia, pinotage (pnt (tq209)). Here we describe a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize haem. The loss of Atpif1 impairs haemoglobin synthesis in zebrafish, mouse and human haematopoietic models as a consequence of diminished Fech activity and elevated mitochondrial pH. To understand the relationship between mitochondrial pH, redox potential, [2Fe-2S] clusters and Fech activity, we used genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, as well as pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to perturbations in Atpif1-regulated mitochondrial pH and redox potential. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize haem, resulting in anaemia. The identification of mitochondrial Atpif1 as a regulator of haem synthesis advances our understanding of the mechanisms regulating mitochondrial haem homeostasis and red blood cell development. An ATPIF1 deficiency may contribute to important human diseases, such as congenital sideroblastic anaemias and mitochondriopathies.

Human mitochondrial ferritin improves respiratory function in yeast mutants deficient in iron-sulfur cluster biogenesis, but is not a functional homologue of yeast frataxin.

We overexpressed human mitochondrial ferritin in frataxin-deficient yeast cells (Δyfh1), but also in another mutant affected in [Fe-S] assembly (Δggc1). Ferritin was correctly processed and expressed in the mitochondria of these cells, but the fraction of total mitochondrial iron bound to ferritin was very low, and most of the iron remained in the form of insoluble particles of ferric phosphate in these mitochondria, as evidenced by gel filtration analysis of the mitochondrial matrix (fast protein liquid chromatography [FPLC]) and by Mössbauer spectroscopy. Mutant cells in which ferritin was overexpressed still accumulated iron in the mitochondria and remained deficient in [Fe-S] assembly, suggesting that human mitochondrial ferritin is not a functional homologue of yeast frataxin. However, the respiratory function was improved in these mutants, which correlates with an improvement of cytochrome and heme synthesis. Overexpression of mitochondrial ferritin in [Fe-S] mutants resulted in the appearance of a small pool of high-spin ferrous iron in the mitochondria, which was probably responsible for the improvement of heme synthesis and of the respiratory function in these mutants.

Regulation of ribonucleotide reductase during iron limitation.

In this issue of Molecular Cell, Sanvisens et al. (2011) report a new mechanism for regulation of yeast ribonucleotide reductase activity that occurs during iron deprivation.

Co-precipitation of phosphate and iron limits mitochondrial phosphate availability in Saccharomyces cerevisiae lacking the yeast frataxin homologue (YFH1).

Saccharomyces cerevisiae cells lacking the yeast frataxin homologue (Δyfh1) accumulate iron in the mitochondria in the form of nanoparticles of ferric phosphate. The phosphate content of Δyfh1 mitochondria was higher than that of wild-type mitochondria, but the proportion of mitochondrial phosphate that was soluble was much lower in Δyfh1 cells. The rates of phosphate and iron uptake in vitro by isolated mitochondria were higher for Δyfh1 than wild-type mitochondria, and a significant proportion of the phosphate and iron rapidly became insoluble in the mitochondrial matrix, suggesting co-precipitation of these species after oxidation of iron by oxygen. Increasing the amount of phosphate in the medium decreased the amount of iron accumulated by Δyfh1 cells and improved their growth in an iron-dependent manner, and this effect was mostly transcriptional. Overexpressing the major mitochondrial phosphate carrier, MIR1, slightly increased the concentration of soluble mitochondrial phosphate and significantly improved various mitochondrial functions (cytochromes, [Fe-S] clusters, and respiration) in Δyfh1 cells. We conclude that in Δyfh1 cells, soluble phosphate is limiting, due to its co-precipitation with iron.

Evidence that yeast frataxin is not an iron storage protein in vivo.

Yeast cells deficient in the yeast frataxin homolog (Yfh1p) accumulate iron in their mitochondria. Whether this iron is toxic, however, remains unclear. We showed that large excesses of iron in the growth medium did not inhibit growth and did not decrease cell viability. Increasing the ratio of mitochondrial iron-to-Yfh1p by decreasing the steady-state level of Yfh1p to less than 100 molecules per cell had very few deleterious effects on cell physiology, even though the mitochondrial iron concentration greatly exceeded the iron-binding capacity of Yfh1p in these conditions. Mössbauer spectroscopy and FPLC analyses of whole mitochondria or of isolated mitochondrial matrices showed that the chemical and biochemical forms of the accumulated iron in mitochondria of mutant yeast strains (Deltayfh1, Deltaggc1 and Deltassq1) displayed a nearly identical distribution. This was also the case for Deltaggc1 cells, in which Yfh1p was overproduced. In these mitochondria, most of the iron was insoluble, and the ratio of soluble-to-insoluble iron did not change when the amount of Yfh1p was increased up to 4500 molecules per cell. Our results do not privilege the hypothesis of Yfh1p being an iron storage protein in vivo.

Friedreich ataxia: molecular mechanisms, redox considerations, and therapeutic opportunities.

Mitochondrial dysfunction and oxidative damage are at the origin of numerous neurodegenerative diseases like Friedreich ataxia and Alzheimer and Parkinson diseases. Friedreich ataxia (FRDA) is the most common hereditary ataxia, with one individual affected in 50,000. This disease is characterized by progressive degeneration of the central and peripheral nervous systems, cardiomyopathy, and increased incidence of diabetes mellitus. FRDA is caused by a dynamic mutation, a GAA trinucleotide repeat expansion, in the first intron of the FXN gene. Fewer than 5% of the patients are heterozygous and carry point mutations in the other allele. The molecular consequences of the GAA triplet expansion is transcription silencing and reduced expression of the encoded mitochondrial protein, frataxin. The precise cellular role of frataxin is not known; however, it is clear now that several mitochondrial functions are not performed correctly in patient cells. The affected functions include respiration, iron-sulfur cluster assembly, iron homeostasis, and maintenance of the redox status. This review highlights the molecular mechanisms that underlie the disease phenotypes and the different hypothesis about the function of frataxin. In addition, we present an overview of the most recent therapeutic approaches for this severe disease that actually has no efficient treatment.

Overexpression of the yeast frataxin homolog (Yfh1): contrasting effects on iron-sulfur cluster assembly, heme synthesis and resistance to oxidative stress.

Friedreich's ataxia is generally associated with defects in [Fe-S] cluster assembly/stability and heme synthesis and strong susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model of Friedreich's ataxia to study the physiological consequences of modulating the expression of the frataxin gene (YFH1). We show that the number of frataxin molecules per wild-type cell varies from less than 200 to 1500 according to the iron concentration in the medium. Cells overexpressing YFH1 on a plasmid (2muYFH1; about 3500 molecules Yfh1/cell) took up more iron than wild-type cells and displayed defective [Fe-S] cluster assembly/stability in vivo. By contrast, endogenous mitochondrial iron was more available to ferrochelatase in 2muYFH1 cells than in wild-type cells, resulting in higher levels of heme synthesis in vitro. Frataxin overproduction resulted in a shift from frataxin trimers to frataxin oligomers of higher molecular mass in the mitochondrial matrix. Much fewer carbonylated proteins were present in 2muYFH1 cells, and these cells were more resistant to oxidizing agents than wild-type cells, which probably resulted from the lower production of hydrogen peroxide by the mitochondria of 2muYFH1 cells compared to wild-type cells. To our knowledge, this work is the first description where major frataxin-related phenotypes ([Fe-S] cluster assembly and heme synthesis) can be split in vivo, suggesting that frataxin has independent roles in both processes, and that the optimal conditions for these independent roles are different.