Cytochrome c oxidase isoform IV‐2 is involved in 3‐nitropropionic acid‐induced toxicity in striatal astrocytes

Abstract

Astrocyte mitochondria play an important role for energy supply and neuronal survival in the brain. Toxic and degenerative processes are largely associated with mitochondrial dysfunction. We, therefore, investigated the effect of 3-nitropropionic acid (NPA), a mitochondrial toxin and in vitro model of Huntington's disease (HD), on mitochondrial function and viability of primary striatal astrocytes. Although NPA is known as an irreversible inhibitor of succinate dehydrogenase, we observed an increase of astrocyte ATP levels after NPA treatment. This effect could be explained by NPA-mediated alterations of cytochrome c oxidase subunit IV isoform (COX IV) expression. The up-regulation of COX isoform IV-2 caused an increased enzyme activity at the expense of elevated mitochondrial peroxide production causing increased cell death. The application of a small interfering RNA against COX IV-2 revealed the causal implication of COX isoform IV-2 in NPA-mediated elevation of oxidative stress and necrotic cell death. Thus, we propose a novel, additional mechanism of NPA-induced cell stress and death which is based on structural and functional changes of astrocyte COX and which could indirectly impair neuronal survival.

Brain region-specific vulnerability of astrocytes in response to 3-nitropropionic acid is mediated by cytochrome c oxidase isoform expression

“…Forward and reverse primers for specific amplification of cox4i1 (5′-TATGCTTTCCCCACT TACGC-3′ and 5′-GCCCACAACTGTCTTCCATT-3′) and cox4i2 (5′-AGATGAACCATCGCTCCAAC-3′ and 5′-ATGGGGTTGCTCTTCATGTC-3′) were designed to eliminate the possibility of amplifying genomic DNA (Singh et al 2009). After reverse transcription, a standard cDNA dilution series and sample cDNA diluted 1:10 were added to a solution containing 5 µM primers and IQ SYBR Green Supermix (Bio-Rad) consisting of 25 U/ml iTaq polymerase, 50 mM KCl, 20 mM TRIS-HCl, 0.2 mM each dNTP, 3 mM MgCl 2 , SYBR Green I, and stabilizers.…”

“…The luminescent reaction during absorbance/illumination at 562 nm was analyzed immediately in a microplate reader (Victor, 1420 Multilabel Counter, Perkin Wallac GmbH, Freiburg, Germany). The ATP content of the NPA-treated cells was calculated and normalized to the amount of proteins determined by applying the BCA Protein Assay Kit (Pierce, Bonn, Germany) with respect to untreated controls set as 100% (Singh et al 2009). …”

“…Freshly isolated mitochondria from astrocytes (as described in detail in Singh et al 2009) were resuspended in a minimal volume of mitochondrial buffer and added to 100 µl Krebs-Ringer phosphate buffer composed of 145 mM NaCl, 5.7 mM NaH 2 PO 4 , 4.86 mM KCl, 0.54 mM CaCl 2 , 1.22 mM MgSO 4 , 5.5 mM glucose, pH 7.35, containing 50 µM Amplex Red reagent and 0.1 U/ml horseradish peroxidase. Hydrogen peroxide in mitochondrial samples was detected by the formation of resorufin, the fluorescent oxidation product of Amplex Red, by using a fluorescence microplate reader (Tecan GENios, Crailsheim, Germany).…”

“…COX IV-2 is only marginally expressed in brain tissue and in astrocytes under physiological conditions. However, under hypoxic and toxic conditions, COX isoform IV-2 is up-regulated (Horvat et al 2006;Singh et al 2009). Cortical astrocytes exposed to hypoxia, i.e., conditions that are equivalent to depriving COX of its catalytic substrate, showed an up-regulation of COX IV-2.…”

“…Each monomer of the dimeric enzyme complex consists of three mitochondriaencoded catalytic and ten nucleus-encoded regulatory subunits (Tsukihara et al 1996;Grossman and Lomax 1997). The nucleus-encoded COX subunit IV-1 plays a crucial role in regulating energy metabolism and oxidative stress Kadenbach et al 2000Kadenbach et al , 2004Horvat et al 2006;Singh et al 2009). At high intracellular energy levels, ATP binds to the N-terminus of COX subunit IV-1 and inhibits the enzyme activity allosterically Kadenbach 1997, 1999;Horvat et al 2006).…”