iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease

Mutations in PANK2 lead to neurodegeneration with brain iron accumulation. PANK2 has a role in the biosynthesis of coenzyme A (CoA) from dietary vitamin B5, but the neuropathological mechanism and reasons for iron accumulation remain unknown. In this study, atypical patient-derived fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently differentiated into cortical neuronal cells for studying disease mechanisms in human neurons. We observed no changes in PANK2 expression between control and patient cells, but a reduction in protein levels was apparent in patient cells. CoA homeostasis and cellular iron handling were normal, mitochondrial function was affected; displaying activated NADH-related and inhibited FADH-related respiration, resulting in increased mitochondrial membrane potential. This led to increased reactive oxygen species generation and lipid peroxidation in patient-derived neurons. These data suggest that mitochondrial deficiency is an early feature of the disease process and can be explained by altered NADH/FADH substrate supply to oxidative phosphorylation. Intriguingly, iron chelation appeared to exacerbate the mitochondrial phenotype in both control and patient neuronal cells. This raises caution for the use iron chelation therapy in general when iron accumulation is absent

Comparison between the HCV IRES domain IV RNA structure and the Iron Responsive Element

Background: Serum ferritin and hepatic iron concentrations are frequently elevated in patients who are chronically infected with the hepatitis C virus (HCV), and hepatic iron concentration has been used to predict response to interferon therapy, but these correlations are not well understood. The HCV genome contains an RNA structure resembling an iron responsive element (IRE) in its internal ribosome entry site (IRES) structural domain IV (dIV). An IRE is a stem loop structure used to control the expression of eukaryotic proteins involved in iron homeostasis by either inhibiting ribosomal binding or protecting the mRNA from nuclease degradation. The HCV structure, located within the binding site of the 40S ribosomal subunit, might function as an authentic IRE or by an IRE-like mechanism.----- Results: Electrophoretic mobility shift assays showed that the HCV IRES domain IV structure does not interact with the iron regulatory protein 1 (IRP1) in vitro. Systematic HCV IRES RNA mutagenesis suggested that IRP1 cannot accommodate the shape of the wild type HCV IRES dIV RNA structure.----- Conclusion The HCV IRES dIV RNA structure is not an authentic IRE. The possibility that this RNA structure is responsible for the observed correlations between intracellular iron concentration and HCV infection parameters through an IRE-like mechanism in response to some other cellular signal remains to be tested

Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications.

Methylation of cytosine deoxynucleotides generates 5-methylcytosine (m(5)dC), a well-established epigenetic mark. However, in higher eukaryotes much less is known about modifications affecting other deoxynucleotides. Here, we report the detection of N(6)-methyldeoxyadenosine (m(6)dA) in vertebrate DNA, specifically in Xenopus laevis but also in other species including mouse and human. Our methylome analysis reveals that m(6)dA is widely distributed across the eukaryotic genome and is present in different cell types but is commonly depleted from gene exons. Thus, direct DNA modifications might be more widespread than previously thought.M.J.K. was supported by the Long-Term Human Frontiers Fellowship (LT000149/2010-L), the Medical Research Council grant (G1001690), and by the Isaac Newton Trust Fellowship (R G76588). The work was sponsored by the Biotechnology and Biological Sciences Research Council grant BB/M022994/1 (J.B.G. and M.J.K.). The Gurdon laboratory is funded by the grant 101050/Z/13/Z (J.B.G.) from the Wellcome Trust, and is supported by the Gurdon Institute core grants, namely by the Wellcome Trust Core Grant (092096/Z/10/Z) and by the Cancer Research UK Grant (C6946/A14492). C.R.B. and G.E.A. are funded by the Wellcome Trust Core Grant. We are grateful to D. Simpson and R. Jones-Green for preparing X. laevis eggs and oocytes, F. Miller for providing us with M. musculus tissue, T. Dyl for X. laevis eggs and D. rerio samples, and to Gurdon laboratory members for their critical comments. We thank U. Ruether for providing us with M. musculus kidney DNA (Entwicklungs- und Molekularbiologie der Tiere, Heinrich Heine Universitaet Duesseldorf, Germany). We also thank J. Ahringer, S. Jackson, A. Bannister and T. Kouzarides for critical input and advice, M. Sciacovelli and E. Gaude for suggestions.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nsmb.314

Variation of selfing rate and inbreeding depression among individuals and across generations within an admixed Cedrus population

[EN] We investigated the variation and short-term evolution of the selfing rate and inbreeding depression (ID) across three generations within a cedar forest that was established from admixture ca 1860. The mean selfing rate was 9.5%, ranging from 0 to 48% among 20 seed trees (estimated from paternally inherited chloroplast DNA). We computed the probability of selfing for each seed and we investigated ID by comparing selfed and outcrossed seeds within progenies, thus avoiding maternal effects. In all progenies, the germination rate was high (88-100%) and seedling mortality was low (0-12%). The germination dynamics differed significantly between selfed and outcrossed seeds within progenies in the founder gene pool but not in the following generations. This transient effect of selfing could be attributed to epistatic interactions in the original admixture. Regarding the seedling growth traits, the ID was low but significant: 8 and 6% for height and diameter growth, respectively. These rates did not vary among generations, suggesting minor gene effects. At this early stage, outcrossed seedlings outcompeted their selfed relatives, but not necessarily other selfed seedlings from other progenies. Thus, purging these slightly deleterious genes may only occur through within-family selection. Processes that maintain a high level of genetic diversity for fitness-related traits among progenies also reduce the efficiency of purging this part of the genetic load. © 2011 Macmillan Publishers Limited All rights reserved. Guardar / Salir Siguiente >This work has been partially supported by Grant PPI-00-04 from the Polytechnic University of Valencia (Spain). We thank B Fady and E Klein as well as two anonymous reviewers for their helpful comments on a previous version of the paper. We acknowledge B Jouaud, W Brunetto, F Jean and H Picot for seed collection and processing and laboratory assistance, as well as P Brahic and staff from the Experimental Nursery of Aix-Les Milles for nursery cares.Ferriol Molina, M.; Pichot, C.; Lefevre, F. (2011). Variation of selfing rate and inbreeding depression among individuals and across generations within an admixed Cedrus population. Heredity. 106(1):146-157. https://doi.org/10.1038/hdy.2010.451461571061Barret SH, Eckert CG (1990). Variation and evolution of mating systems in seed plants. In: Kawano S (ed). Biological Approaches and Evolutionary Trends in Plants. Academic Press: London. pp 230–254.Benton TG, Plaistow SJ, Coulson TN (2006). Complex population dynamics and complex causation: devils, details and demography. Proc R Soc B Biol Sci 273: 1173–1181.Bower AD, Aitken SN (2007). Mating system and inbreeding depression in whitebark pine (Pinus albicaulis Engelm.). Tree Genet Genomes 3: 379–388.Byers DL, Waller DM (1999). Do plant populations purge their genetic load? Effects of population size and mating history on inbreeding depression. Annu Rev Ecol Syst 30: 479–513.Cointat M (1996). Le roman du cèdre. Revue Forestière Française 48: 503–526.Collevatti RG, Grattapaglia D, Duvall J (2001). High resolution microsatellite based analysis of the mating system allows the detection of significant biparental inbreeding in Caryocar brasiliense, an endangered tropical tree species. Heredity 86: 60–67.Cottrell JE, White IMS (1995). The use of isozyme genetic markers to estimate the rate of outcrossing in a Sitka pruce (Picea sitchensis (Bong.) Carr.) seed orchard in Scotland. New Forests 10: 111–122.Coulson T, Benton TG, Lundberg P, Dall SRX, Kendall BE (2006). Putting evolutionary biology back in the ecological theatre: a demographic framework mapping genes to communities. Evol Ecol Res 8: 1155–1171.Durel CE, Bertin P, Kremer A (1996). Relationship between inbreeding depression and inbreeding coefficient in maritime pine (Pinus pinaster). Theor Appl Genet 92: 347–356.Eriksson E (2006). Thinning operations and their impact on biomass production in stands of Norway spruce and Scots pine. Biomass Bioenergy 30: 848–854.Fady B, Lefèvre F, Reynaud M, Vendramin GG, Bou Dagher-Karrat M, Anzidei M et al. (2003). Gene flow among different taxonomic units: evidence from nuclear and cytoplasmic markers in Cedrus plantation forests. Theor Appl Genet 107: 1132–1138.Farris MA, Mitton JB (1984). Population density, outcrossing rate, and heterozygote superiority in ponderosa pine. Evolution 38: 1151–1154.Favre-Duchartre M (1970). Des Ovules Aux Graines. Monographie 8. Masson et Cie.: Paris.Franklin EC (1969). Inbreeding Depression in Metrical Traits of Loblolly Pine (Pinus taeda L.) as a Result of Self-pollination. North Carolina State University: Raleigh, NC. Technical report No 40, School of Forest Resources.Gregorius HR, Ziehe M, Ross MD (1987). Selection caused by self-fertilization I. Four measures of self-fertilization and their effects on fitness. Theor Popul Biol 31: 91–115.Hamrick JL, Godt MJ (1989). Allozyme diversity in plant species. In: Brown AHD, Al Kahler MC, Weir BS (eds). Plant Population Genetics, Breeding, and Genetic Resources. Sinauer: Sunderland, MA. pp 43–63.Holsinger KE (1991). Mass-action models of plant mating systems—the evolutionary stability of mixed mating systems. Am Nat 138: 606–622.Husband BC, Schemske DW (1996). Evolution of the magnitude and timing of inbreeding depression in plants. Evolution 50: 54–70.Jones FA, Hamrick JL, Peterson CJ, Squiers ER (2006). Inferring colonization history from analyses of spatial genetic structure within populations of Pinus strobus and Quercus rubra. Mol Ecol 15: 851–861.Kärkkäinen K, Savolainen O (1993). The degree of early inbreeding depression determines the selfing rate at the seed stage: model and results from Pinus sylvestris (Scots pine). Heredity 71: 160–166.Keller LF, Waller DM (2002). Inbreeding effects in wild populations. Trends Ecol Evol 17: 230–241.Klein EK, Lavigne C, Gouyon PH (2006). Mixing of propagules from discrete sources at long distance: comparing an exponential tail to an exponential. BMC Ecol 6: 3.Knowles P, Furnier GR, Aleksiuk MK, Perry DJ (1987). Significant levels of self-fertilization in natural populations of tamarack. Can J Bot 65: 1087–1091.Koelewijn HP, Koski V, Savolainen O (1999). Magnitude and timing of inbreeding depression in Scots pine (Pinus sylvestris L.). Evolution 53: 758–768.Kremer A (1994). Genetic diversity and phenotypic variability of forest trees. Genet Sel Evol 26: s105–s123.Krouchi F, Derridj A, Lefèvre F (2004). Year and tree effect on reproductive organisation of Cedrus atlantica in a natural forest. For Ecol Manage 197: 181–189.Lande R (1988). Genetics and demography in biological conservation. Science 241: 1455–1460.Ledig FT (1986). Heterozygosity, heterosis, and fitness in outbreeding plants. In: Soulé ME (ed). Conservation Biology: the Science of Scarcity and Diversity. Sinauer Ass: Sunderland. pp 77–104.Lee JK, Nordheim EV, Kang H (1996). Inference for lethal gene estimation with application in plants. Biometrics 52: 451–462.Lefèvre F, Fady B, Fallour-Rubio D, Ghosn D, Bariteau M (2004). Impact of founder population, drift and selection on the genetic diversity of a recently translocated tree population. Heredity 93: 542–550.Marquardt PE, Epperson BK (2004). Spatial and population genetic structure of microsatellites in white pine. Mol Ecol 13: 3305–3315.Morgante M, Vendramin GG, Rossi P (1991). Effects of stand density on outcrossing rate in two Norway spruce (Picea abies) populations. Can J Bot 69: 2704–2708.Mosseler A, Major JE, Simpson JD, Daigle B, Lange K, Park YS et al. (2000). Indicators of population viability in red spruce, Picea rubens. I. Reproductive traits and fecundity. Can J Bot 78: 928–940.Naydenov KD, Tremblay FM, Alexandrov A, Fenton NJ (2005). Structure of Pinus sylvestris L. populations in Bulgaria revealed by chloroplast microsatellites and terpenes analysis : provenance tests. Biochem Syst Ecol 33: 1226–1245.Neale DB, Adams WT (1985). The mating system in natural and shelterwood stands of Douglas-fir. Theor Appl Genet 71: 201–207.Notivol E, Garcia-Gil MR, Alia R, Savolainen O (2007). Genetic variation of growth rhythm traits in the limits of a latitudinal cline in Scots pine. Can J For Res 37: 540–551.O’Connell LM, Russell J, Ritland K (2004). Fine-scale estimation of outcrossing in western redcedar with microsatellite assay of bulked DNA. Heredity 93: 443–449.Parducci L, Szmidt AE, Madaghiele A, Anzidei M, Vendramin GG (2001). Genetic variation at chloroplast microsatellites (CpSSRs) in Abies nebrodensis (Lojac.) Mattei and three neighboring Abies species. Theor Appl Genet 102: 733–740.Parraguirre-Lezama C, Vargas-Hernández JJ, Ramirez-Vallejo P, Ramirez Herrera C (2004). Mating system in four natural populations of Pinus greggii Engelm. Agrociencia 38: 107–119.Petit RJ, Hampe A (2006). Some evolutionary consequences of being a tree. Annu Rev Ecol Evol Syst 37: 187–214.Pichot C, Bastien C, Courbet F, Demesure-Musch B, Dreyfus P, Fady B et al. (2006). Déterminants et conséquences de la qualité génétique des graines et semis lors de la phase initiale de régénération naturelle des peuplements forestiers. In: 6e Colloque National du BRG ; La Rochelle 2006/10/02-04. Les Actes du Bureau des Ressources Génétiques 6: 277–297.Remington DL, O’Malley DM (2000a). Whole-genome characterization of embryonic stage inbreeding depression in a selfed loblolly pine family. Genetics 155: 337–348.Remington DL, O’Malley DM (2000b). Evaluation of major genetic loci contributing to inbreeding depression for survival and early growth in a selfed family of Pinus taeda. Evolution 54: 1580–1589.Restoux G, Silva DE, Sagnard F, Torre F, Klein E, Fady B (2008). Life at the margin: the mating system of Mediterranean conifers. Web Ecol 8: 94–102.Ribeiro MM, Mariette S, Vendramin GG, Szmidt AE, Plomion C, Kremer A (2002). Comparison of genetic diversity estimates within and among populations of maritime pine using chloroplast simple-sequence repeat and amplified fragment length polymorphism data. Mol Ecol 11: 869–877.Ritland K, El-Kassaby YA (1985). The nature of inbreeding in a seed orchard of Douglas fir as shown by an efficient multi-locus model. Theor Appl Genet 71: 375–384.Ritland K, Travis S (2004). Inferences involving individual coefficients of relatedness and inbreeding in natural populations of Abies. For Ecol Manage 197: 171–180.Robledo-Arnuncio JJ, Alia R, Gil L (2004). Increased selfing and correlated paternity in a small population of a predominantly outcrossing conifer, Pinus sylvestris. Mol Ecol 13: 2567–2577.Rouault G, Turgeon J, Candau JN, Roques A, Aderkas P (2004). Oviposition strategies of conifer seed chalcids in relation to host phenology. Naturwissenschaften 91: 472–480.Savolainen O, Kärkkäinen K, Kuittinen H (1992). Estimating numbers of embryonic lethals in conifers. Heredity 69: 308–314.Scofield DG, Schultz ST (2006). Mitosis, stature and evolution of plant mating systems: low-Phi and high-Phi plants. Proc R Soc B Biol Sci 273: 275–282.Shaw DV, Allard RW (1982). Estimation of outcrossing rates in douglas-fir using isoenzyme markers. Theor Appl Genet 62: 113–120.Skrøppa T (1996). Diallel crosses in Picea abies. II. Performance and inbreeding depression of selfed families. For Genet 3: 69–79.Sorensen FC (1997). Effects of sib mating and wind pollination on nursery seedling size, growth components, and phenology of Douglas-fir seed-orchard progenies. Can J For Res 27: 557–566.Sorensen FC (1999). Relationship between self-fertility, allocation of growth, and inbreeding depression in three coniferous species. Evolution 53: 417–425.Sorensen FC (2001). Effect of population outcrossing rate on inbreeding depression in Pinus contorta var. murrayana seedlings. Scand J For Res 16: 391–403.Sorensen FC, Adams WT (1993). Self fertility and natural selfing in three Oregon Cascade populations of lodgepole pine. In: Lindgren D (ed). Pinus contorta—From Untamed Forest to Domesticated Crop. Department of Forest Genetics and Plant Physiology, Sweden University of Agricultural Science: Umea, Sweden. Report 11, pp 358–374.Sorensen FC, Miles RS (1974). Self-pollination effects on Douglas fir and ponderosa pine seeds and seedlings. Silvae Genet 23: 135–138.Sorensen FC, Miles RS (1982). Inbreeding depression in height, height growth, and survival of Douglas-fir, ponderosa pine, and noble fir to 10 years of age. For Sci 28: 283–292.Terrab A, Paun O, Talavera S, Tremetsberger K, Arista M, Stuessy TF (2006). Genetic diversity and population structure in natural populations of Moroccan Atlas cedar (Cedrus atlantica; Pinaceae) determined with cpSSR markers. Am J Bot 93: 1274–1280.Vendramin GG, Lelli L, Rossi P, Morgante M (1996). A set of primers for the amplification of 20 chloroplast microsatellites in Pinaceae. Mol Ecol 5: 595–598.White TL, Adams WT, Neale DB (2007). Forest Genetics. CABI Publisher: Cambridge, MA. pp 149–186.Wilcox MD (1983). Inbreeding depression and genetic variances estimated from self- and cross- pollinated families of Pinus radiata. Silvae Genet 32: 89–96.Williams CG (2007). Re-thinking the embryo lethal system within the Pinaceae. Can J Bot 85: 667–677.Williams CG (2008). Selfed embryo death in Pinus taeda: a phenotypic profile. New Phytol 178: 210–222.Williams CG, Auckland LD, Reynolds MM, Leach KA (2003). Overdominant lethals as part of the conifer embryo lethal system. Heredity 91: 584–592.Wilson R (1923). Life history of Cedrus atlantica. Bot Gaz 75: 203–208.Yazdani R, Muona O, Rudin D, Szmidt AE (1985). Genetic structure of a Pinus sylvestris L. seed-tree stand and naturally regenerated understory. For Sci 31: 430–436

Ceruloplasmin Deficiency Reduces Levels of Iron and BDNF in the Cortex and Striatum of Young Mice and Increases Their Vulnerability to Stroke

Ceruloplasmin (Cp) is an essential ferroxidase that plays important roles in cellular iron trafficking. Previous findings suggest that the proper regulation and subcellular localization of iron are very important in brain cell function and viability. Brain iron dyshomeostasis is observed during normal aging, as well as in several neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases, coincident with areas more susceptible to insults. Because of their high metabolic demand and electrical excitability, neurons are particularly vulnerable to ischemic injury and death. We therefore set out to look for abnormalities in the brain of young adult mice that lack Cp. We found that iron levels in the striatum and cerebral cortex of these young animals are significantly lower than wild-type (WT) controls. Also mRNA levels of the neurotrophin brain derived neurotrophic factor (BDNF), known for its role in maintenance of cell viability, were decreased in these brain areas. Chelator-mediated depletion of iron in cultured neural cells resulted in reduced BDNF expression by a posttranscriptional mechanism, suggesting a causal link between low brain iron levels and reduced BDNF expression. When the mice were subjected to middle cerebral artery occlusion, a model of focal ischemic stroke, we found increased brain damage in Cp-deficient mice compared to WT controls. Our data indicate that lack of Cp increases neuronal susceptibility to ischemic injury by a mechanism that may involve reduced levels of iron and BDNF

A Machine Learning Approach for Identifying Novel Cell Type–Specific Transcriptional Regulators of Myogenesis

Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA–based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. We first assembled a small number of enhancers that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, we increased the number of enhancers by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. We then built and trained a classifier using this enhancer set and identified additional related enhancers based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, we found extensive diversity in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type–specific developmental gene expression patterns

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference