Title: A non‐enzymatic function of 17β‐hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival
Abstract: Research Article4 February 2010Open Access A non-enzymatic function of 17β-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival Katharina Rauschenberger Katharina Rauschenberger Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Katja Schöler Katja Schöler Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Jörn Oliver Sass Jörn Oliver Sass Laboratory of Clinical Biochemistry and Metabolism, Freiburg University Children's Hospital, Freiburg, Germany Search for more papers by this author Sven Sauer Sven Sauer Department of Paediatrics I, Heidelberg University, Heidelberg, Germany Search for more papers by this author Zdenka Djuric Zdenka Djuric Department of Medicine I and Clinical Chemistry, Heidelberg University, Heidelberg, Germany Search for more papers by this author Cordula Rumig Cordula Rumig Molecular Immunology, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Nicole I. Wolf Nicole I. Wolf Department of Paediatrics V, Heidelberg University, Heidelberg, Germany Department of Child Neurology, VU University Medical Center, Amsterdam, The Netherlands Search for more papers by this author Jürgen G. Okun Jürgen G. Okun Department of Paediatrics I, Heidelberg University, Heidelberg, Germany Search for more papers by this author Stefan Kölker Stefan Kölker Department of Paediatrics I, Heidelberg University, Heidelberg, Germany Search for more papers by this author Heinz Schwarz Heinz Schwarz Microscopy Unit, Max-Planck-Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Christine Fischer Christine Fischer Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Beate Grziwa Beate Grziwa Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Heiko Runz Heiko Runz Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Astrid Nümann Astrid Nümann Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Naeem Shafqat Naeem Shafqat Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Kathryn L. Kavanagh Kathryn L. Kavanagh Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Günter Hämmerling Günter Hämmerling Molecular Immunology, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Ronald J. A. Wanders Ronald J. A. Wanders Department of Paediatrics and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Julian P. H. Shield Julian P. H. Shield Royal Hospital for Children, Bristol, UK Search for more papers by this author Udo Wendel Udo Wendel University Children's Hospital, Düsseldorf, Germany Search for more papers by this author David Stern David Stern College of Medicine, University of Cincinnati, Cincinnati, OH, USA Search for more papers by this author Peter Nawroth Peter Nawroth Department of Medicine I and Clinical Chemistry, Heidelberg University, Heidelberg, Germany Search for more papers by this author Georg F. Hoffmann Georg F. Hoffmann Department of Paediatrics I, Heidelberg University, Heidelberg, Germany Search for more papers by this author Claus R. Bartram Claus R. Bartram Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Bernd Arnold Bernd Arnold Molecular Immunology, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Angelika Bierhaus Angelika Bierhaus Department of Medicine I and Clinical Chemistry, Heidelberg University, Heidelberg, Germany Search for more papers by this author Udo Oppermann Udo Oppermann Botnar Research Center, Oxford Biomedical Research Unit; University of Oxford, Oxford, UK Search for more papers by this author Herbert Steinbeisser Herbert Steinbeisser Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Johannes Zschocke Corresponding Author Johannes Zschocke [email protected] Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Divisions of Human Genetics and Clinical Genetics, Medical University Innsbruck, Innsbruck, Austria Search for more papers by this author Katharina Rauschenberger Katharina Rauschenberger Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Katja Schöler Katja Schöler Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Jörn Oliver Sass Jörn Oliver Sass Laboratory of Clinical Biochemistry and Metabolism, Freiburg University Children's Hospital, Freiburg, Germany Search for more papers by this author Sven Sauer Sven Sauer Department of Paediatrics I, Heidelberg University, Heidelberg, Germany Search for more papers by this author Zdenka Djuric Zdenka Djuric Department of Medicine I and Clinical Chemistry, Heidelberg University, Heidelberg, Germany Search for more papers by this author Cordula Rumig Cordula Rumig Molecular Immunology, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Nicole I. Wolf Nicole I. Wolf Department of Paediatrics V, Heidelberg University, Heidelberg, Germany Department of Child Neurology, VU University Medical Center, Amsterdam, The Netherlands Search for more papers by this author Jürgen G. Okun Jürgen G. Okun Department of Paediatrics I, Heidelberg University, Heidelberg, Germany Search for more papers by this author Stefan Kölker Stefan Kölker Department of Paediatrics I, Heidelberg University, Heidelberg, Germany Search for more papers by this author Heinz Schwarz Heinz Schwarz Microscopy Unit, Max-Planck-Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Christine Fischer Christine Fischer Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Beate Grziwa Beate Grziwa Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Heiko Runz Heiko Runz Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Astrid Nümann Astrid Nümann Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Naeem Shafqat Naeem Shafqat Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Kathryn L. Kavanagh Kathryn L. Kavanagh Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Günter Hämmerling Günter Hämmerling Molecular Immunology, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Ronald J. A. Wanders Ronald J. A. Wanders Department of Paediatrics and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Julian P. H. Shield Julian P. H. Shield Royal Hospital for Children, Bristol, UK Search for more papers by this author Udo Wendel Udo Wendel University Children's Hospital, Düsseldorf, Germany Search for more papers by this author David Stern David Stern College of Medicine, University of Cincinnati, Cincinnati, OH, USA Search for more papers by this author Peter Nawroth Peter Nawroth Department of Medicine I and Clinical Chemistry, Heidelberg University, Heidelberg, Germany Search for more papers by this author Georg F. Hoffmann Georg F. Hoffmann Department of Paediatrics I, Heidelberg University, Heidelberg, Germany Search for more papers by this author Claus R. Bartram Claus R. Bartram Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Bernd Arnold Bernd Arnold Molecular Immunology, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Angelika Bierhaus Angelika Bierhaus Department of Medicine I and Clinical Chemistry, Heidelberg University, Heidelberg, Germany Search for more papers by this author Udo Oppermann Udo Oppermann Botnar Research Center, Oxford Biomedical Research Unit; University of Oxford, Oxford, UK Search for more papers by this author Herbert Steinbeisser Herbert Steinbeisser Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Search for more papers by this author Johannes Zschocke Corresponding Author Johannes Zschocke [email protected] Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Divisions of Human Genetics and Clinical Genetics, Medical University Innsbruck, Innsbruck, Austria Search for more papers by this author Author Information Katharina Rauschenberger1, Katja Schöler1,2, Jörn Oliver Sass3, Sven Sauer4, Zdenka Djuric5, Cordula Rumig6, Nicole I. Wolf7,8, Jürgen G. Okun4, Stefan Kölker4, Heinz Schwarz9, Christine Fischer1, Beate Grziwa1, Heiko Runz1, Astrid Nümann1, Naeem Shafqat2, Kathryn L. Kavanagh2, Günter Hämmerling6, Ronald J. A. Wanders10, Julian P. H. Shield11, Udo Wendel12, David Stern13, Peter Nawroth5, Georg F. Hoffmann4, Claus R. Bartram1, Bernd Arnold6, Angelika Bierhaus5, Udo Oppermann14, Herbert Steinbeisser1 and Johannes Zschocke *,1,15 1Institute of Human Genetics, Heidelberg University, Heidelberg, Germany 2Structural Genomics Consortium, University of Oxford, Oxford, UK 3Laboratory of Clinical Biochemistry and Metabolism, Freiburg University Children's Hospital, Freiburg, Germany 4Department of Paediatrics I, Heidelberg University, Heidelberg, Germany 5Department of Medicine I and Clinical Chemistry, Heidelberg University, Heidelberg, Germany 6Molecular Immunology, German Cancer Research Center, Heidelberg, Germany 7Department of Paediatrics V, Heidelberg University, Heidelberg, Germany 8Department of Child Neurology, VU University Medical Center, Amsterdam, The Netherlands 9Microscopy Unit, Max-Planck-Institute for Developmental Biology, Tübingen, Germany 10Department of Paediatrics and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 11Royal Hospital for Children, Bristol, UK 12University Children's Hospital, Düsseldorf, Germany 13College of Medicine, University of Cincinnati, Cincinnati, OH, USA 14Botnar Research Center, Oxford Biomedical Research Unit; University of Oxford, Oxford, UK 15Divisions of Human Genetics and Clinical Genetics, Medical University Innsbruck, Innsbruck, Austria *Tel: +43 512 9003-70500; Fax: +43 512 9003 73510 EMBO Mol Med (2010)2:51-62https://doi.org/10.1002/emmm.200900055 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease-causing gene is HSD17B10 and encodes 17β-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10−/− mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine-restricted diet are required. The paper explained PROBLEM: HSD10 is an essential enzyme in the isoleucine breakdown pathway and has also been reported as an important mediator of mitochondrial toxicity in Alzheimer's disease. A deficiency of HSD10 caused by mutations in the HSD17B10 gene can be recognized by specific metabolic changes. However, children with HSD10 deficiency show a neurodegenerative disease picture that does not resemble other disorders of isoleucine metabolism or similar organic acidurias but is more reminiscent of primary mitochondrial disorders. The exact pathomechanism is unknown. RESULTS: By investigating additional patients with a genetic deficiency of HSD10 we show that there is no correlation between enzyme activity and clinical presentation. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10−/− mice demonstrate that HSD10 is essential for structural and functional integrity of mitochondria independently of its enzymatic activity. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. IMPACT: The exact molecular mechanisms leading to mitochondrial disintegration and neuronal apoptosis in HSD10 deficiency are still unknown but our data show that the clinical effects cannot be attributed to the accumulation of toxic metabolites in the isoleucine pathway or other metabolic effects. Rather, HSD10 has a protective effect on mitochondrial integrity. Delineation of this protective mechanism should provide new therapeutic perspectives for HSD10 dysfunction. Children with a genetic deficiency of HSD10 are unlikely to benefit from an isoleucine-restricted diet, previously suggested as a therapeutic option. Treatment should aim at reducing mitochondrial stress and maintaining mitochondrial homeostasis through proactive management of infections and fever and possibly the administration of vitamins and cofactors. INTRODUCTION Organic acidurias are inherited metabolic disorders caused by the deficiency of enzymes involved in the mitochondrial oxidation of coenzyme A (CoA)-activated acyl compounds derived from amino acid breakdown. Clinical symptoms frequently develop at times of increased protein catabolism due to fasting or illness, leading to increased flux through oxidative pathways and accumulation of pathological metabolites. We have previously reported a novel organic aciduria caused by a deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) involved in isoleucine metabolism (Zschocke et al, 2000). The clinical picture of this condition is very different from other organic acidurias. Most of the affected children do not develop metabolic crises and show few symptoms in the first year of life. From the second year on they follow a neurodegenerative disease course associated with mitochondrial dysfunction, leading to progressive loss of skills, neurological abnormalities including epilepsy, cardiomyopathy, retinal degeneration and death. The causative HSD17B10 gene is located on the X-chromosome and encodes 17β-hydroxysteroid dehydrogenase type 10 (HSD17B10, abbreviated HSD10) (Ofman et al, 2003). Several studies have indicated that this protein (also known as ERAB = endoplasmatic reticulum-associated amyloid-β-binding protein, or ABAD = amyloid-β-binding alcohol dehydrogenase) binds amyloid-β (Aβ) and mediates mitochondrial toxicity in Alzheimer's disease (AD, Lustbader et al, 2004; Yan et al, 1997). HSD10 is thus implicated in two neurodegenerative disorders but the pathogenetic basis has so far remained elusive. Here we report that the severity of symptoms in patients with MHBD deficiency (MHBDD) is not correlated with residual enzyme activity of the mutated HSD10 proteins, suggesting that the HSD10 protein has other functions in addition to the enzymatic activity. In vertebrate systems, no loss-of-function data are available for HSD10, so far. We present evidence in Xenopus and mouse embryos, as well as in fibroblasts from patients with MHBDD that HSD10 is required for structural and functional integrity of the mitochondria. This function is independent of the enzymatic activity of the protein. Rescue experiments with wild type (WT) and mutant protein in Xenopus embryos in which HSD10 function was knocked-down revealed striking functional differences in properties of mutations found in MHBDD patients. Mutation Q165H, associated with complete loss of enzymatic activity but normal neurological development in humans, prevented apoptosis induced by HSD10 knock-down. In contrast, no rescue of the apoptosis phenotype was observed with human mutations associated with classical disease presentation (R130C, up to 64% residual enzyme activity) or a very severe clinical phenotype (D86G, 30% residual enzyme activity). These results were corroborated by rescue experiments in dendritic cells derived from homozygous HSD10 Tie2-Cre knock-out mice and indicated that the enzymatic activity of HSD10 is not required for cell survival. Our data indicate that loss of (non-enzymatic) HSD10 function mediated by gene mutation, knock-down or knock-out causes mitochondrial dysfunction and apoptotic cell death. These findings shed new light on the pathogenesis of the clinical features of MHBDD and argue for new approaches to therapy. They also may be of relevance to the understanding of neurodegeneration in other conditions. RESULTS Enzymatic activity of mutated HSD10 protein and severity of symptoms in patients do not correlate In order to gain insight into the pathogenesis of neurodegeneration in MHBD deficiency and to obtain further information on the biological role of HSD10, we investigated additional patients with MHBDD. The condition was retrospectively diagnosed in one child (case 1, Supporting Information) with a very severe neonatal presentation, absent neurological development and death from progressive hypertrophic cardiomyopathy at the age of 7 months. MHBD activity in fibroblasts, as determined in two independent laboratories, was only partially reduced to approximately 30% of normal. This was considerably higher than for other patients with MHBDD. Sequence analysis of the complete coding region of the HSD17B10 gene in this patient revealed hemizygosity for a novel mutation p.D86G (c.257A>G) in exon 3; no other mutation was identified. MHBDD was also diagnosed in a boy (case 2, Supporting Information) who presented with pre- and postnatal failure to thrive but normal cognitive and motor development. Neurological examination in this boy and two affected relatives (brother and cousin) has been entirely normal up to the present age of 8 years. There was no measurable MHBD activity in fibroblasts; molecular studies identified hemizygosity for the novel mutation p.Q165H (c.495A>C) in exon 5 of the HSD17B10 gene. Observations in these patients indicated that the development and severity of symptoms in MHBDD is unrelated to residual enzyme activity. In order to confirm these findings we carried out in vitro expression analyses of WT HSD10 and the relevant mutations R130C (the common mutation), D86G (associated with a very severe clinical phenotype) and Q165H (associated with normal development). WT and mutants were expressed as stable, soluble proteins (Supporting Information Fig 1). Further insights were gained through the 3D structure determination of human HSD10 (Fig 1A, B), which crystallized in spacegroup P3221 and was determined to high resolution (1.2 Å; data collection and refinement details in Supporting Information Table 1). Kinetic constants were determined with different substrates (Fig 1E, F). Kinetic parameters calculated for the WT enzyme were in line with earlier data reported for this enzyme (Shafqat et al, 2003). Residual dehydrogenase activities of 64 and 28% were measured for mutations R130C and D86G, respectively; however, mutation R130C was unstable at room temperature and steadily lost enzyme activity (Fig 1C). Protein stability was assessed with and without NAD+ or NADH cofactor as a function of temperature by differential scanning fluorimetry (Fig 1D). Cofactor binding of NAD+ or NADH, as reflected by increased thermal stability, was observed for both WT and D86G. In contrast, mutation Q165H located at the active centre of the enzyme showed neither residual activity nor cofactor binding in our experimental settings. Inability of the mutation Q165H to bind the cofactor indicates that regardless of the substrate, no enzymatic reaction requiring NADH or NAD+ (all known reactions) will be sufficiently catalysed by this mutant protein. WT and all mutations alike showed identical mitochondrial distribution (data not shown). The lack of correlation between disease severity and residual enzyme activity in MHBDD patients thus is not due to misdirection of intracellular transport or impaired mitochondrial localization of mutated HSD10. Mitotracker staining revealed a filamentous network-like structure of the mitochondria in WT cells and fibroblasts carrying the Q165H mutation. Cells with the R130C and D86G mutations showed punctate and fragmented mitochondrial organization (Fig 1G–J). Despite the differences in the Mitotracker staining the amount of mitochondrial material is not reduced in patient cells judged by the amount of mitochondrial marker Grp75 protein and TOMM20 mRNA (Supporting Information Fig 2). The amount of HSD10 transcripts was nearly identical in the cells analysed (WT, R130C and Q165H). In agreement with the finding that the R130C mutation is unstable (Fig 1C) less HSD10 protein could be detected in fibroblasts carrying this mutation (Supporting Information Fig 2). Figure 1. Crystal structure, stability and activity of the HSD10 homotetramer and mitochondrial morphology in fibroblasts from MHBDD patients. Crystal structure of HSD10. (A) Diagram of the homotetramer. A NADH cofactor is bound to one monomer. (B) Diagram of a HSD10 dimer with the location of the mutations. The active centre is marked by the substrate in red. C.. Enzyme activity of HSD10 over time. Kcat/KM of HSD10 WT and mutations measured in a 30 min interval; substrate 2-methyl-3-hydroxybutyryl-CoA. D.. Stability of HSD10 WT and mutations. Mean value of Tm determined by differential scanning fluorimetry (DSF)-experiments and standard deviation are shown. Black column: no cofactor/substrate, grey column: cofactor NAD+, white column: cofactor NADH (* indicates significant cofactor binding). E, F.. Enzyme activity of HSD10 with different substrates. (E) Kcat/KM of HSD10 mutations with hydroxybutyryl-CoA as substrate (WT enzyme is taken as 100%). (F) Kcat/KM of HSD10 mutations with 2-methyl-3-hydroxybutyryl-CoA as substrate in relativity to the WT enzyme. G–J.. Mitochondrial staining in patient fibroblasts. 300 nM Mitotracker Green FM were used on cells fixed with 3.7% formaldehyde on coverslips and mitochondria were visualized on a Perkin Elmer spinning disc confocal ERS-FRET on Nikon TE2000 inverted microscope. (G) Control, (H) R130C, (I) D86G, (J) Q165H. K.. Fibroblasts were sectioned for electron microscopy. Pictures of 10–43 random systematically chosen visual fields were taken in a magnification of 11.5 × 103, scale bars: 100 nm. Mitochondria were classified into three groups (1—dense, dark; 2—loosely packed; 3—depleted cristae) with group 3 not only being morphologically distinct but also characterized by smaller mitochondria. Total numbers per sample and an overview of the cells are given in Supporting Information Fig 3. ** Indicate significance at p < 0.0001, * gives significance at p = 0.0366–0.0857 adjusted for multiple comparisons within the experiment compared to control fibroblasts. Normal human dermal fibroblasts served as controls. Download figure Download PowerPoint Mutations D86G and R130C cause severe disruption of mitochondrial morphology Since HSD10 WT and mutations are localized to mitochondria and patients with MHBDD show signs of mitochondrial dysfunction, we analysed mitochondrial morphology by EM in fibroblasts derived from patients compared with control fibroblasts (Fig 1K and Supporting Information Fig 3). The majority of mitochondria in control cells showed WT morphology (dense, dark). Fibroblasts carrying the Q165H mutation maintained WT morphology of 45% of the mitochondria, 30% displayed an intermediate phenotype with loosely packed and/or swollen cristae and 27% showed depletion of cristae and appeared 'empty' (for details, see Material and Methods). In contrast, in cells carrying the D86G and R130C mutation 65–85% of the mitochondria displayed an aberrant phenotype (group 2 and 3). The EM analysis of mitochondria of HSD10 mutant human cells thus showed that HSD10 is required for mitochondrial integrity. This function is not correlated with residual enzyme activity. Mitochondrial disintegration after conditional Hsd17b10 knock-out in mice Since knock-out of Hsd17b10 in mice results in early embryonic lethality at gastrula stages (B. Arnold, unpublished data), we established two conditional Hsd17b10 knock-out mouse lines. In one line, Hsd17b10 was eliminated in endothelial cells and cells of the immune system by mating mice carrying a floxed allele of Hsd17b10 with a Tie2 promoter driven Cre recombinase strain. These mice are viable and fertile, but have defects in spleen and vasculature. The animals die rapidly around week 25. Supporting Information Fig 4 shows the recombination efficiency in spleen cells and dendritic cells of Tie2-Cre × Hsd17b10 mice as an example. To generate the second conditional knock-out line, mice carrying a floxed allele of Hsd17b10 were mated with a DBH-Cre line which eliminates the Hsd17b10 gene in noradrenergic neurons. These mice are viable and fertile but die around week 26. In the central nervous system (CNS) the locus coeruleus contains noradrenergic neurons and we set out to analyse the morphology of the mitochondria in this region by EM in conditional knock-out mice. In the loci coerulei of DBH-Cre HSD10 deficient mice, almost 30% of the mitochondria showed depletion of cristae and appeared 'empty', more than 50% of the mitochondria were loosely packed and had swollen cristae, while normal morphology (dense, dark) was only found in 20% (Fig 2, Supporting Information Fig 5). In the cerebellum of the same mice, which lacks noradrenergic neurons, 50% of the mitochondria had WT morphology, 40% displayed the intermediate phenotype and only 10% appeared 'empty' and without cristae. A similar picture was observed in the peripheral nervous system (PNS). Mitochondria were morphologically severely altered in superior cervical ganglia isolated from DBH-Cre conditional knock-out mice (Supporting Information Figs 6 and 7). The EM analysis of two independent primary cell types (neurons of the CNS and PNS) from Hsd17b10 DBH-Cre conditional knock-out mice thus confirmed in a genetic system that HSD10 is required for mitochondrial structural integrity in the CNS and the PNS and that the observed changes in mitochondria are cell type independent. Figure 2. Mitochondrial morphology in brains of mice with a conditional knock-out in noradrenergic neurons (DBH-Cre). Brains from knock-out mice were Vibratome sectioned (50 µm), the loci coerulei, which could be identified by pigmentation, were dissected and prepared for electron microscopy. Scale bar: 100 µm. Mitochondria were classified into three groups (1—dense, dark; 2—loosely packed/swollen cristae; 3—depleted cristae). Total numbers per sample and an overview of the cells are given in Supporting Information Fig 5. Sections of the cerebellum, which lacks noradrenergic neurons, served as a control. * Indicates significance (p < 0.0001) of differences to the cerebellum. Pictures of 33 random systematically chosen visual fields were taken in a magnification of 11.5 × 103, scale bars: 100 nm. Download figure Download PowerPoint HSD10 knock-down in Xenopus impairs mitochondrial integrity and induces apoptosis In order to study HSD10 loss of function in early vertebrate embryos we turned to Xenopus as an experimental model. We cloned the Xenopus homologue of HSD10 (xHSD10) and performed RT-PCR analysis, as well as whole mount in situ hybridization during the first 3 days of embryonic development. HSD10 mRNA is provided maternally, and zygotic HSD10 transcription was found from neurula stages onward (Supporting Information Fig 8). In Xenopus embryos, translation of mRNAs can be specifically blocked by Morpholino (Mo) antisense oligonucleotides. Two Mo antisense oligonucleotides were designed to inhibit HSD10 mRNA translation. Mo5′UTR targeted the 5′UTR and MoATG the coding region of xHSD10. Both oligos specifically suppressed HSD10 translation in Xenopus embryos (Fig 3A). Figure 3. Mitochondrial function and morphology in Xenopus animal caps. A.. Functionality and specificity of antisense Mo oligonucleotides is shown. Antisense Mo oligonucleotides and Myc-tagged HSD10 cDNA were injected in Xenopus embryos. Protein extract from these embryos was subjected to Western blot using 9E10 anti-Myc antibody. Antisense Mo oligonucleotide Mo5′UTR can block the translation of 5′xHSD10 cDNA including the 5′UTR, but not the translation of ATGxHSD10 cDNA starting with the start codon. MoATG can block translation of both constructs