Rybacka-Mossakowska 2017 MiP2017

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Mitochondrial respiration in intact peripheral blood mononuclear cells in multiple sclerosis patients.

Link: MiP2017

Rybacka-Mossakowska J, Michalak S, Osztynowicz K, Biernacka-Lukanty J, Tokarz-Kupczyk E, Kozubski W (2017)

Event: MiP2017

COST Action MITOEAGLE
Traditionally, multiple sclerosis (MS) is considered an autoimmune, inflammatory disease [1]. However, accumulating evidence indicates a role of neurodegeneration in the pathogenesis of MS [2]. Neuroapthological features of central nervous system (CNS) lesions during MS are well characterized and include: breakdown of the blood-brain barrier, multifocal inflammation, demyelination, oligodendrocyte loss, reactive gliosis, and axonal degeneration [3]. It has not been fully explained whether microglia or monocyte-derived macrophages contribute to neurodegeneration proces. Chronic inflammation in MS may trigger neurodegeneration through the production of reactive oxigen species (ROS) and reactive nitrogen species (RNS), mitochondrial injury, metabolic stress, protein misfolding and a loss of neurones [4,5]. Such a hypothesis is supported by clinical observation of limited effect of available immunomodulatory drugs on long-term MS progression. The aim of this study was to evaluate mitochondrial respiration in intact peripheral blood mononuclear cells (PBMCs) in MS patients.

We enrolled in the study 13 multiple sclerosis patients hospitalized in Department of Neurology at Poznan University of Medical Sciences in Poznan, Poland and 23 healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA blood via density gradient centrifugation (Histopaque, Sigma-Aldrich). The isolated fractions were supplemented with protease inhibitors cocktail (Sigma-Aldrich; 1:200 vol/vol) on ice. The cell number was counted in Bürker’s chamber and the volume corresponding to 1x106 cells was applied for respirometry. Cell viability was tested using the trypan blue method. Mitochondrial respiration was analyzed in intact PBMCs according to the ROUTINE, LEAK, electron transfer-pathway (ET-pathway), and residual oxygen consumption (ROX) protocol [6] using a High-Resolution FluoRespirometer (Oxygraph-2k; Oroboros Instruments, Innsbruck, Austria). Briefly, a total of 1x106 PBMCs were added to the respirometer chamber after 10 min of stabilization at 37 °C and incubated with continuous stirring at a speed of 750 rpm. The data were subsequently collected with the application of DatLab software 6.1.0.7 (Oroboros Instruments, Innsbruck, Austria).

ROUTINE respiration in PBMCs from heparinized blood was lower in MS patients (12.66 ± 3.77 pmol O2/s*106 cells, mean ± standard deviation, P = 0,0480) than in controls (15.76 ± 4.54 pmol O2/s*106 cells). No changes in LEAK respiration were found in MS patients (4.25 ± 2.06 pmol O2/s*106 cells, P = 0.1431) compared to healthy subjects (4.52± 1.77 pmol O2/s*106 cells). ET-pathway respiration in PBMCs from MS patients (14.44; 10.46 – 22.94 pmol O2/s*106 cells, median; interquartile range) did not differ from controls (16.54; 11.38 – 20.68 pmol O2/s*106 cells). Downregulation of ROX respiration was observed in PBMCs from MS patients (2.90 ± 1.70 pmol O2/s*106 cells) compared to controls (4.76 ±1.79 pmol O2/s*106 cells, P = 0.0004).

Basic mitochondrial respiration is downregulated in peripheral blood mononuclear cells in the course of multiple sclerosis. The inhibition of oxidative side reactions may result from modification of the activity of key regulatory enzymes by specific immunoglobulins.


Bioblast editor: Kandolf G O2k-Network Lab: PL Poznan Michalak S


Labels: MiParea: Respiration, Patients  Pathology: Neurodegenerative 

Organism: Human  Tissue;cell: Blood cells  Preparation: Intact cells 


Coupling state: LEAK, ROUTINE, ET  Pathway: ROX  HRR: Oxygraph-2k 

PMBCs 

Affiliations

Rybacka–Mossakowska J(1), Slawomir Michalak(1), Osztynowicz K(1), Biernacka – Lukanty J(1), Tokarz–Kupczyk E(2), Kozubski W(2)
  1. Dept Neurochem Neuropathol
  2. Dept Neurology; Poznan Univ Medical Sciences, Poznan, Poland. – swami@ump.edu.pl


References

  1. Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG (2000) Multiple sclerosis. N Engl J Med 343:938-52.
  2. Dutta R, Trapp BD (2011) Mechanisms of neuronal dysfunction and degeneration in Multiple Sclerosis. Prog Neurobiol 93: 1–12.
  3. Prineas J (2001) Pathology of Multiple Sclerosis. In: Cook, S., editor. Handbook of Multiple Sclerosis. Marcel Dekker 2001:289-324.
  4. Fischer MT, Sharma R, Lim JL, Haider L, Frischer JM, Drexhage J, Mahad D, Bradl M, van Horssen J, Lassmann H (2012) NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury. Brain 135:886–99.
  5. Haider L, Fischer MT, Frischer JM, Bauer J, Höftberger R, Botond G, Esterbauer H, Binder CJ, Witztum JL, Lassmann H (2011) Brain. 134:1914-24.
  6. Gnaiger E, Renner-Sattler K (2014) High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ET-pathway, ROX. Mitochondrial Physiology Network Vol 08.09:1–8.