Letm1, the mitochondrial Ca2+/H+ antiporter, is PNAS PLUS essential for normal glucose metabolism and alters brain function in Wolf–Hirschhorn syndrome Dawei Jianga,b,c, Linlin Zhaoa,b,c, Clary B. Clishd, and David E. Claphama,b,c,1 aDepartment of Cardiology, Howard Hughes Medical Institute, bManton Center for Orphan Disease, Children’s Hospital Boston, and cDepartment of Neurobiology, Harvard Medical School, Boston, MA 02115; and dBroad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02142 Contributed by David E. Clapham, May 6, 2013 (sent for review February 18, 2013) Mitochondrial metabolism, respiration, and ATP production neces- we generated Letm1-deficient mice and found that Letm1 knock- 2+ 2+ sitate ion transport across the inner mitochondrial membrane. down reduced Ca mito uptake at low [Ca ]cyto,impairedmito- Leucine zipper-EF-hand containing transmembrane protein 1 (Letm1), chondrial ATP generation capacity, disrupted early embryonic one of the genes deleted in Wolf–Hirschhorn syndrome, encodes development, altered glucose metabolism, and increased suscepti- a putative mitochondrial Ca2+/H+ antiporter. Cellular Letm1 knock- bility to seizures. These results are consistent with the WHS seizure 2+ + down reduced Ca mito uptake, H mito extrusion and impaired mi- phenotype in humans and show that Letm1 alters mitochondrial tochondrial ATP generation capacity. Homozygous deletion of energetic performance and metabolic pathways. Letm1 in mice resulted in embryonic lethality before day 6.5 of embryogenesis and ∼50% of the heterozygotes died before day Results 13.5 of embryogenesis. The surviving heterozygous mice exhibited 2+ + Letm1 Regulates [Ca ]mito and [H ]mito Homeostasis and Mitochondrial altered glucose metabolism, impaired control of brain ATP levels, Energetics. In initial experiments, Letm1 was transiently reduced and increased seizure activity. We conclude that loss of Letm1 con- using siRNAs to investigate short-term responses to protein loss tributes to the pathology of Wolf–Hirschhorn syndrome in humans and mitigate long-term developmental or compensatory mecha- PHYSIOLOGY and may contribute to seizure phenotypes by reducing glucose nisms. Letm1 knockdown by RNA interference in Flp-In-293 cells oxidation and other specific metabolic alterations. 2+ 2+ resulted in dramatically reduced Ca mito uptake in low [Ca ]cyto + + that was coupled with H mito extrusion (Fig. 1A). Na -dependent calcium signaling | mitochondria | epilepsy 2+ + 2+ Ca mito extrusion, mediated by the Na /Ca exchanger, balances 2+ 2+ steady-state [Ca ]mito levels in low [Ca ]cyto; a dramatically re- itochondria are major effectors and regulators of in- 2+ 2+ duced steady-state [Ca ]mito was measured in Letm1 knockdown Mtracellular [Ca ]. Calcium-mediated signal transduction cells in the presence of 10 mM Na+. In contrast, high [Ca2+] cyto across the inner mitochondrial membrane (IMM) links in- 2+ triggered a fast [Ca ]mito rise in cells with Letm1 knockdown to creased metabolic demand to ATP production rate because A 2+ a level similar to that in controls (Fig. 1 ). These results suggest Ca regulates key metabolic enzymes, metabolite trans- 2+ 2+ + 2+ that Letm1 is crucial for Ca mito homeostasis in low [Ca ]cyto. porters, and the F1F0 H -ATPase (1, 2). Excessive Ca ac- Constant H+ pumping by the electron transport chain (ETC) ΔΨ cumulation reduces mitochondrial membrane potential ( mito), establishes a highly negative membrane potential across the impairs ATP production, precipitates phosphate, and triggers cell 2+ fi mitochondrial inner membrane. Cells in which Letm1 was death. Ca extrusion across the IMM represents a signi cant knocked down (Fig. 1B) resulted in similar or slightly higher energy cost at normal ΔΨmito (∼−180 mV) and thus mitochon- 2+ 2+ tetramethylrhodamine methyl ester (TMRM) accumulation (Fig. drial Ca (Ca mito) signaling is tightly controlled under physi- 1C), consistent with minimal changes in mitochondrial mem- ological conditions (3–5). brane potential. Certain populations of mitochondria with in- The mitochondrial Ca2+ uniporter (MCU), a highly Ca2+- 2+ creased volume can be identified in Letm1 knockdown cells, but selective channel (6), dominates fast Ca mito uptake when [Ca2+] is high, significantly buffers extramitochondrial Ca2+,and cyto Significance depolarizes ΔΨmito (7, 8). Because the MCU’s Vmax is orders of 2+ magnitude higher than that of Ca mito exchange mechanisms, 2+ 2+ – repetitive high Ca cyto elevations trigger Ca mito overload and Wolf Hirschhorn syndrome (WHS), caused by deletions in can lead to the mitochondrial permeability transition (9, 10). Free 4p16.3, was the first recognized subtelomeric deletion syn- 2+ drome. As with other syndromes of this class, WHS has not yet [Ca ]mito in intact cells usually fluctuates below the micromolar range, suggesting that Ca2+ exchangers are crucial for maintaining been subjected to an intensive, systematic analysis using mouse 2+ 2+ or cellular models. We recently identified leucine zipper-EF-hand Ca mito homeostasis at low Ca cyto levels to preserve mitochon- drial homeostasis and bioenergetics. The Na+/Ca2+ exchanger, containing transmembrane protein 1 (Letm1) as a mitochondrial 2+ + Ca2+/H+ antiporter, and potentially others, control Ca2+ across Ca /H antiporter, located in the core WHS region. In this study, the IMM. Mitochondrial transporters and channels are rapidly Letm1 null mice were generated and characterized. Deletion of being identified (11). Letm1 disrupts early embryonic development, reduces glucose We previously performed a genome-wide RNAi screen in oxidation and thus particularly affects brain function. Drosophila S2 cells that identified Letm1 (leucine zipper-EF- hand containing transmembrane protein 1) as a mitochondrial Author contributions: D.J. and D.E.C. designed research; D.J. and L.Z. performed research; 2+ + D.J., L.Z., and C.B.C. contributed new reagents/analytic tools; D.J., L.Z., and C.B.C. analyzed Ca /H antiporter (12). Letm1 is an evolutionarily conserved, data; and D.J., C.B.C., and D.E.C. wrote the paper. ubiquitously expressed IMM protein. Heterozygous deletion of The authors declare no conflict of interest. Letm1 – a chromosomal region containing is associated with Wolf 1To whom correspondence should be addressed. E-mail: [email protected]. Hirschhorn syndrome (WHS), a disease characterized by cranio- edu. facial defects, growth and mental retardation, muscle hypotonia, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. congenital heart defects, and seizures (13). In the current study, 1073/pnas.1308558110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1308558110 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 A D suggesting that homologous deletion of Letm1 is embryonically lethal (Fig. S1C). Mendelian ratios of littermate genotypes in- +/− dicated that approximately half of the Letm1 mice died before +/− birth. We isolated and analyzed embryos from Letm1 inter- crosses at various times after fertilization and determined that −/− Letm1 embryos died before day 6.5 of embryogenesis (E6.5) +/− and ∼50% of the Letm1 embryos died before E13.5 (Fig. 2A). These results suggest a Letm1 gene dosage effect on early em- bryonic development and survival. +/− E9.5 Letm1 embryos were growth-retarded (Fig. 2B), with Flp-In-293 a reduced rate of DNA synthesis (BrdU incorporation), but exhibited no significant increase in apoptosis (Fig. 2C). De- veloping embryos use significantly more ATP in support of rapid growth and differentiation; [ATP] in E9.5 embryos was ∼10-fold higher than in adult differentiated tissues (Fig. 2D). We specu- +/− late that early Letm1 embryogenesis is compromised due to reduced ATP generation capacity, a defect with variable pene- E trance that can be partially compensated by glycolysis. After the critical stages of early embryogenesis, surviving E13.5 +/− Letm1 embryos seem grossly normal (Fig. 3A). Mitochondrial morphology and ΔΨmito in cultured primary fibroblasts from +/− B kDa Letm1 embryos were similar to WT (Fig. 3 B–D). Primary +/− 2+ 80 fibroblasts from Letm1 embryos had reduced Ca mito uptake Letm1 2+ rates, low steady-state [Ca ]mito, and reduced matrix pH com- A 40 pared with WT (Fig. S2 ). O2 consumption rates were similar in Actin fibroblasts grown in high-glucose media with pyruvate supplement siRNA: Control Letm1 C F 100 A 75 50 25 Flp-In-293 Cell number % of control 0 Day 0 Day 3 Day 6 2+ + Fig. 1. Letm1 knockdown reduces [Ca ]mito and [H ]mito, but has minor 2+ + effects on ΔΨmito.(A)[Ca ]mito and [H ]mito measured by mt-pericam in digitonin-permeabilized Flp-In-293 cells treated with control (n = 4) or Letm1 +/- +/+ +/- siRNAs (n = 4) for 4 d. Traces shown are the mean mitochondrial signals from B C the whole imaging field. Ca2+ and Na+ were applied as indicated. (B) Letm1 protein levels in Flp-In-293 cells treated with control siRNAs (left two lanes) or Letm1 siRNAs (right three lanes from three independent experiments) analyzed by Western blotting. (C) Plots of TMRM measurements of ΔΨmito in control and Letm1 siRNA-treated Flp-In-293 cells (mean ± SD). (D) Images showing TMRM-labeled mitochondria following siRNA treatment for 4 d. +/+ Blue arrowheads denote enlarged mitochondria. (Scale bar, 2 μm.) (E) [ATP] +/+ +/- of FLP-In-293 cells treated with Letm1 siRNA for 4 d normalized to control D cells (n = 3). (F) Percent of surviving FLP-In-293 cells treated with Letm1 siRNA for 6 d normalized