MiP2005: Session 9

Mitochondrial Physiology Network 10.9: 106 (2005) - download pdf

 

A study of cytotoxicity of phytanic acid in mitochondria and astrocytes isolated from rat brain – possible mechanism in Refsum disease.

Peter Schönfeld1, S Kahlert2, G Reiser2

1Inst. Biochemistry; 2Inst. Neurobiochemistry, Otto-von-Guericke-University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany. - peter.schoenfeld@medizin.uni-magdeburg.de

    In Refsum disease, a peroxisomal genetic disorder, the branched-chain fatty acid phytanic acid (3,7,11,15-tetramethylhexadecanoic acid; Phyt) accumulates at high levels (up to 1 mM) throughout the body. Clinical features suggest that Phyt exerts severe cytotoxicity, mostly in tissues with a high energy turnover. We studied the influence of non-esterified Phyt on various parameters of the energy metabolism in rat brain by using isolated mitochondria and brain cells.

    Single-cell analysis applied to isolated hippocampal astrocytes reveals that Phyt (100 µM) drastically increases the inflow of extracellular Ca2+, exerts strong depolarization of mitochondria in situ. Furthermore, cell viability of cultured astrocytes was significantly reduced after a 5h-exposure to Phyt. All these changes were not seen with the unbranched palmitic acid.

    Isolated mitochondria become strongly deenergized by Phyt, applied at low concentrations (5 – 20 µM, i.e. 5 – 20 nmol/mg of mitochondrial protein). In energized mitochondria, deenergization is mainly due to protonophoric activity of Phyt. In addition, Phyt decreases state 3 respiration by inhibition of the electron flow within the respiratory chain and inhibition of the ADP/ATP exchange across the inner membrane.

    As an important functional consequence of these findings, mitochondria preloaded with small amounts of Ca2+ (100 nmol/mg protein) become highly sensitized to rapid membrane permeability transition (MPT), even when only low concentrations of Phyt (below 5 µM) are applied. Depolarisation of the inner mitochondrial membrane and locking the ADP/ATP carrier in the matrix conformation most likely account for this sensitization to MPT.

    Moreover, the interaction of Phyt with components of the respiratory chain raises strongly mitochondrial superoxide generation (O2·-), an observation which is not seen with palmitic acid. Interaction of Phyt with complex I mostly contributes to Phyt-related O2·-  generation. This conclusion is supported by (i) inhibition of NADH-ubiquinone oxidoreductase and (ii) decreased reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In addition, similar to antimycin A (complex III-inhibitor), Phyt increases complex III-related O2·- generation.

    In conclusion, the observed harmful effects of Phyt on brain mitochondria and on astrocytes support the hypothesis that clinical features of Refsum disease are directly related to pathologically increased levels of Phyt.

1.    Wanders RJA, Jakobs C, Skjeldal OH (2001) Refsum disease. In: The metabolic and molecular bases of inherited disease. 8th ed (Scriver CR, Beaudet AL, Sly WS, Valle D, eds), McGraw-Hill, New York: 3303-3321.

2.    Schönfeld P, Kahlert S, Reiser G (2004) In brain mitochondria the branched-chain fatty acid phytanic acid impairs energy transduction and sensitizes for permeability transition. Biochem. J. 383: 121-128.

3.  Kahlert S, Schönfeld P, Reiser G (2005) The Refsum disease marker phytanic acid, a branched chain fatty acid, affects Ca2+ homeostasis and mitochondria, and reduces cell viability in rat hippocampal astrocytes. Neurobiol. Dis. 18: 110-118.


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