XB-ART-41002EMBO Mol Med February 1, 2010; 2 (2): 51-62.
A non-enzymatic function of 17beta-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival.
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 17beta-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.
PubMed ID: 20077426
PMC ID: PMC3377269
Article link: EMBO Mol Med
Genes referenced: dbh fadd fas foxg1 hsd17b10 kit myc odc1 otx2 tek
GO keywords: isoleucine metabolic process
Morpholinos: hsd17b10 MO1 hsd17b10 MO2
Disease Ontology terms: organic acidemia
OMIMs: ALZHEIMER DISEASE; AD
Article Images: [+] show captions
|Figure 3. Mitochondrial function and morphology in Xenopus animal capsA. 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.B. Effect of HSD10 knock-down on mitochondrial function. Animal caps were dissected from injected Xenopus embryos and the turnover of 1-C14 pyruvate was measured. Counts per minute (CPM) is shown in relation to uninjected control (100%) and the standard error is given.C, D. Animal caps were dissected from injected Xenopus embryos and sectioned for electron microscopy. Pictures of 24 random systematically chosen visual fields were taken in a magnification of 6.6 × 103, scale bars: 100 nm (C). Mitochondria were classified into three groups (1—dense, dark; 2—loosely packed; 3—depleted cristae) and the distribution is shown in (D). * Indicates significance at p < 0.0001 compared to uninjected control animal caps. Total numbers per sample and an overview of the cells are given in Supporting Information Fig 9.|
|Figure 4. Loss-of-function analysis of xHSD10 using Mo antisense oligonucleotidesA. The phenotype of embryos injected with xHSD10 MoATG and its rescue with hHSD10 are shown. The injection of MoATG resulted in small or no eyes and reduced anterior structures in 99% (n = 60) of the injected embryos compared with control embryos. Differences in pigmentation are due to slightly differing developmental stages of the MoATG injected and the rescued embryo. Rescue of this phenotype was shown in two independent experiments (n = 40) where 92% of the embryos were WT and only 8% showed the knock-down phenotype.B, C. The reduction of forebrain and mid-/hindbrain tissue is shown by in situ hybridization of embryos injected with MoATG in comparison to uninjected controls using BF1 and otx2 as a markers. Fourteen out of 15 embryos (otx2) and 13 out of 15 embryos (BF1), respectively, showed reduction of forebrain and mid-/hindbrain tissue but brain patterning is not disturbed.D. The increase of apoptosis after knock-down of xHSD10 is shown by TUNEL staining (arrowheads). Embryos were injected with HSD10 MoATG on the right side (R). The left side (L) served as an internal control.|
|Figure 5. Quantitative analysis of the effect of HSD10 loss-of-function on the apoptosis rate and rescueExperimental scheme.Quantification of TUNEL staining on the right (injected) side of tailbud stage embryos (NF St. 33/34) normalized against the left (control) side. The standard error of four individual experiments (n = 45–52 embryos) is shown. * Denotes significance of mean difference from uninjected control at p < 0.0001.Dendritic cells from WT mice and mice with a conditional knock-out in endothelial cells and haematopoietic stem cells (Tie2) were transfected with plasmids (pT-Rex-DEST30) bearing human HSD10 WT or mutations for 48 h before TUNEL assay was performed. The percentage of TUNEL positive cells of three independent experiments and standard error are shown. Statistical significance (*) of difference from WT cells was determined at p < 0.0001. The increase in apoptosis rate caused by HSD10 knock-down can be rescued by human HSD10 WT and the Q165H mutation but not by R130C and D86G cDNA.|
|Supplementary Figure 8 Expression pattern of HSD10 mRNA. (A) The spatial expression pattern of HSD10 mRNA is shown by wholemount in situ hybridization. Signal is observed in early stages (NF5 to 10.5) and in tadpole stages (NF41) in ventral parts of the somites, neural tube, pronephros and eye. DIG-labelled antisense RNA as a probe for in situ hybridization was synthesized using Digoxygenin RNA labelling Kit (Roche) with pCS2+_myc/xHSD10 digested with SalI as a template. (B) The temporal expression pattern of HSD10 mRNA is shown by reverse transcription PCR analysis. cDNA for reverse transcription PCR was synthesized from total RNA of Xenopus embryos of different NF stages using Revert Aid M-MuLV RT (Fermentas). For the detection of HSD10, the following primer combinations and a standardized PCR protocol with 30 cycles and a T m of 56,3°C were used: HSD10 5’caccctgtcactgctctgaa3’ and 5’catcttggatttgcccaagt3’ and ODC 5’gtcaatgatggagtgtatggatc3’ and 5’tccattccgctctcctgagcac3’. Maternal mRNA for HSD10 exists until stage NF10.5, zygotic expression starts at stage NF19.5.|
|Supplementary Figure 10 Effect of HSD10 loss-of-function on the apoptosis rate in Xenopus embryos. (A) Xenopus embryos were injected on the right side with MoCo, Mo5’UTR and Fadd (Fas-associated protein with death domain) as a positive control, respectively. Embryos were subjected to TUNEL staining and quantified for apoptotic spots on each side. The uninjected (left) side served as an internal control and the injected side (right) was normalized against the internal control. Embryos shown and numbers are exemplarily. Knock-down of xHSD10 causes an increase in apoptosis. (B-E) frontal view of embryos (B) uninjected control (C) MoCo (D) Fadd (E) Mo5’UTR.|
|Supplementary Figure 12 Localization of HSD10 in Xenopus A6 cells. A6 cells were cultivated in DMEM low glucose medium diluted with 15% water at 24°C and 5% CO2. Transient transfection of A6 cells was achieved with pCS2+_myc/xHSD10 or pT-Rex-DEST30 containing cDNA for hHSD10 wt and the mutations R130C, D86G, Q165H, respectively, using lipofectamine (Invitrogen) according to the manufacturer’s instructions. (A-E) Transfected cells were cultured for 48 h, fixed on coverslips with 3.7% formaldehyde and permeabilized with 0.5% Triton X-100 and 3.7% formaldehyde in PBS for 5 min. Unspecific binding sites were blocked with 20% newborn calf serum in IIF buffer (2% newborn calf serum, 2.5% cold water fish gelatine, 10% glycerol, 0.1% Tween-20, 2% normal serum according to the second antibody in PBS). Overexpressed proteins were detected using 9E10 anti-myc antibody and mouse monoclonal anti-HSD10 antibody (Abcam). After incubation with secondary antibody cells were washed and transferred to 200 nM Mitotracker Red 580 (Invitrogen) for 30 min. The cells were then fixed in 3.7% formaldehyde, mounted in Mowiol and observed by fluorescent microscopy (Olympus SZX12). Fluorescent signal of HSD10 is seen as puncta within the cytosol. Visualization of mitochondria using Mitotracker Red 580. Merged images show clear co-localization between Mitotracker Red and HSD10 in A6 cells regardless of the protein being human (wt or mutated) or Xenopus. The anti-HSD10 antibody does not recognize the endogenous Xenopus HSD10 protein but the transfected human proteins. All proteins (wt and mutations) are stably expressed and show a similar cellular distribution pattern.|
|hsd17b10 (hydroxysteroid (17-beta) dehydrogenase 10 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 5, ?????view.|
|hsd17b10 (hydroxysteroid (17-beta) dehydrogenase 10 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, vegetal view.|
|hsd17b10 (hydroxysteroid (17-beta) dehydrogenase 10 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, dorsal view, anterior left.|
|hsd17b10 (hydroxysteroid (17-beta) dehydrogenase 10 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 41, lateral view, anterior left, dorsal up.|