Poster Presentation ASSCR, AGCTS, ISCT ANZ and Friends Joint Scientific Conference 2019

Modelling the mitochondrial disease Sengers syndrome using human embryonic stem cells (#114)

Yau Chung Low 1 2 , Cameron L McKnight 1 2 , Yilin Kang 3 4 , Thomas D Jackson 3 4 , Daniella H Hock 3 4 , David A Stroud 3 4 , David A Elliott 1 2 , Eric G Hanssen 4 , Mike T Ryan 5 , Diana Stojanovski 3 4 , David R Thorburn 1 2 6 , Ann E Frazier 1 2
  1. Department of Paediatrics, The University of Melbourne, Parkville, VIC, Australia
  2. Murdoch Children's Research Institute, Parkville, VIC, Australia
  3. Department of Biochemistry and Molecular Biology , The University of Melbourne, Parkville, VIC, Australia
  4. The Bio21 Molecular Science and Biotechnology Institute, Parkville, VIC, Australia
  5. Department of Biochemistry and Molecular Biology , Monash University, Clayton, VIC, Australia
  6. Victorian Clinical Genetics Services, Parkville, VIC, Australia

Sengers syndrome is a potentially fatal mitochondrial disease characterised by hypertrophic cardiomyopathy, congenital cataracts, lactic acidosis, and exercise intolerance[1]. This disease is caused by mutations in the acylglycerol kinase (AGK) gene[2,3]. The enzymatic activity of AGK functions in mitochondrial lipid metabolism[4,5].More recently, AGK was discovered to be a subunit of the mitochondrial TIM22 protein import complex. AGK stabilises the TIM22 complex and facilitate mitochondrial carrier protein biogenesis independently to its kinase function[6,7].

 

This project aims to model and investigate the molecular and cellular pathomechanisms underlying Sengers syndrome using human embryonic stem cells (hESCs) differentiated to clinically relevant cardiomyocytes.

 

AGK-/- hESCs were generated using CRISPR/Cas9 gene editing technology, and validated for pluripotency and karyotype. Mutants were characterised by genetic (DNA and RNA) and immunoblot experiments. Selected clones were differentiated into cardiomyocytes and further functional analyses performed including calcium imaging and electron microscopy experiments.

 

DNA sequencing and cDNA studies identified multiple hESC clones with AGK mutations causing a frameshift and premature stop codons, or splicing defects. In hESCs with bi-allelic AGK mutations, the TIM22 complex, and subsequently the ATP carrier protein, were not detected by BN-PAGE. Disrupted ATP carrier biogenesis had a negative impact on ATP production in AGK-/- hESCs. Additionally, preliminary results indicate that cardiomyocytes derived from AGK-/- hESCs display an irregular beating pattern and abnormal calcium handling compared to controls. Furthermore, samples analysed by electron microscopy suggest that mutant cardiomyocytes possess disorganised myofibrils.

 

The AGK-/- hESCs have the potential to provide clinically relevant tissue samples for further investigations of disease pathomechanisms, with future experiments including proteomic and lipidomic analyses of mutants. Ultimately, these cells could be used to facilitate pre-clinical studies testing potential treatments for Sengers syndrome.

  1. [1] Sengers, R. C. A., ter Haar, B. G. A., Trijbels, J. M. F., Willems, J. L., Daniels, O., Stadhouders, A. M. Congenital cataract and mitochondrial myopathy of skeletal and heart muscle associated with lactic acidosis after exercise. J. Pediat. 86: 873-880, 1975.
  2. [2] Calvo, S. E., Compton, A. G., Hershman, S. G., Lim, S. C., Lieber, D. S., Tucker, E. J., Laskowski, A., Garone, C., Liu, S., Jaffe, D. B., Christodoulou, J., Fletcher, J. M., Bruno, D. L., Goldblatt, J., DiMauro, S., Thorburn, D. R., Mootha, V. K. Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing. Sci. Transl. Med. 4: 118ra10, 2012. Note: Electronic Article.
  3. [3] Mayr, J. A., Haack, T. B., Graf, E., Zimmermann, F. A., Wieland, T., Haberberger, B., Superti-Furga, A., Kirschner, J., Steinmann, B., Baumgartner, M. R., Moroni, I., Lamantea, E., Zeviani, M., Rodenburg, R. J., Smeitink, J., Strom, T. M., Meitinger, T., Sperl, W., Prokisch, H. Lack of the mitochondrial protein acylglycerol kinase causes Sengers syndrome. Am. J. Hum. Genet. 90: 314-320, 2012.
  4. [4] Waggoner, D. W., Johnson, L. B., Mann, P. C., Morris, V., Guastella, J., Bajjalieh, S. M. MuLK, a eukaryotic multi-substrate lipid kinase. J. Biol. Chem. 279[6] Vukotic, M., Nolte, H., König, T., Saita, S., Ananjew, M., Krüger, M., Tatsuta, T. and Langer, T., 2017. Acylglycerol kinase mutated in Sengers syndrome is a subunit of the TIM22 protein translocase in mitochondria. Molecular cell, 67(3), pp.471-483. : 38228-38235, 2004.
  5. [5] Bektas, M., Payne, S. G., Liu, H., Goparaju, S., Milstien, S., Spiegel, S. A novel acylglycerol kinase that produces lysophosphatidic acid modulates cross talk with EGFR in prostate cancer cells. J. Cell Biol. 169: 801-811, 2005.
  6. [6] Kang, Y., Stroud, D.A., Baker, M.J., De Souza, D.P., Frazier, A.E., Liem, M., Tull, D., Mathivanan, S., McConville, M.J., Thorburn, D.R. and Ryan, M.T., 2017. Sengers syndrome-associated mitochondrial acylglycerol kinase is a subunit of the human TIM22 protein import complex. Molecular cell, 67(3), pp.457-470.
  7. [7] Vukotic, M., Nolte, H., König, T., Saita, S., Ananjew, M., Krüger, M., Tatsuta, T. and Langer, T., 2017. Acylglycerol kinase mutated in Sengers syndrome is a subunit of the TIM22 protein translocase in mitochondria. Molecular cell, 67(3), pp.471-483.
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