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Kidere 2018 MiPschool Tromso E2

From Bioblast
Dita Kidere
Cytoplasmic hybrid cells as a model to characterize the OXPHOS system: control sample with glutamate metabolism defect.

Link: MitoEAGLE

Kidere D, Makrecka-Kuka M, Stavusis J, Lace B, Inashkina I (2018)

Event: MiPschool Tromso-Bergen 2018

COST Action MitoEAGLE

Mitochondrial diseases are heterogenous multisystem organ disorders which can be caused by alterations in both nuclear and mitochondrial DNA (mtDNA). Defects in the oxidative phosphorylation system (OXPHOS) are presented in many cases. Cytoplasmic hybrid (cybrid) cells have the same nuclear background so they are a good model to investigate the influence of mtDNA alterations on cell function [1]. We have been performing molecular diagnostics of the mtDNA for patients with suspected mitochondrial diseases for several years now and recently we started to use cybrid cell models to evaluate the effect of the mtDNA mutations on the cell functions. To evaluate the OXPHOS system functionality of the mutant cell lines, we compare them to the control lines harbouring the same haplogroup. Thus, cells with mutation m.9185T>C in the gene MT-ATP6 belonging to the haplogroup U4d were compared with the two control lines belonging to haplogroups U5b and U5a. During measurements U5a line showed unexpected results for the control line.

Cybrid cell lines were developed using osteosarcoma derived rho zero cells and platelets from patients and healthy individuals. Healthy donors were chosen due to their belonging to the mtDNA haplogroups. All created cell lines were checked for the suspected haplogroup or mutation. Mitochondrial functionality was determined by high resolution respirometry using Oroboros O2k. Respiratory chain complex I โ€“ IV and additionally complex I+III and II+III activities were measured spectrophotometrically in isolated mitochondria as previously described [2] with modifications.

Till now we have made 4 cybrid cell lines harbouring pathogenic mutations and 15 control cell lines with several clones for each. Up to date respirometry measurements have been performed for two mutant and three control cell lines. Surprisingly the clone 1 of U5a cell line showed no effect in O2 flux after addition of glutamate and succinate, though the addition of rotenone induced a significant inhibition of respiration. Additional measurements of respirometry in another clone of this cell line (clone 2) showed declined effect after addition of glutamate, but overreaction after addition of succinate. Respiratory chain complex activities for both clones of the U5a line were within reference intervals, however they showed higher citrate synthase activity compared to the other control lines, thereby, the ratio of complex activities to citrate synthase activity for complexes I, II, IV and II+III were impaired in comparison to references. Full length mtDNA sequencing for the U5a line revealed no pathogenic mutations.

Respirometry results of the control cybrid cell line harbouring U5a haplogroup suggest possible defect in electron transfer system (more likely glutamate metabolism). Further investigations are necessary to specify the causative mechanism. Our results also remind that all control samples should be checked well before including in experiments and sometimes they can reveal unexpected findings.


โ€ข Bioblast editor: Plangger M โ€ข O2k-Network Lab: LV Riga Makrecka-Kuka M


Affiliations

Kidere D(1), Makrecka-Kuka M(2), Stavusis J(1), Lace B(3), Inashkina I(1)

  1. Latvian Biomedical Research Study Centre
  2. Latvian Inst Organic Synthesis; Riga, Latvia
  3. Centre Hospitalier Univ Quรฉbec, Canada. โ€“ [email protected]

References

  1. Wilkins HM, Carl SM, Swerdlow RH (2014) Cytoplasmic hybrid (cybrid) cell lines as a practical model for mitochondriopathies. Redox Biol 2:619-31.
  2. Feichtinger RG, Zimmermann F, Mayr JA, Neureiter D, Hauser-Kronberger C, Schilling FH, Jones N, Sperl W, Kofler B (2010) Low aerobic mitochondrial energy metabolism in poorly- or undifferentiated neuroblastoma. BMC Cancer 10:149.


Labels: MiParea: Respiration, mtDNA;mt-genetics 


Organism: Human  Tissue;cell: Platelet  Preparation: Isolated mitochondria 


Pathway: N, S, NS  HRR: Oxygraph-2k  Event: E2