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Loss of PLA2G6 leads to elevated mitochondrial lipid peroxidation and mitochondrial dysfunction.

Kinghorn KJ, Castillo-Quan JI, Bartolome F, Angelova PR, Li L, Pope S, Cochemé HM, Khan S, Asghari S, Bhatia KP, Hardy J, Abramov AY, Partridge L - Brain (2015)

Bottom Line: Furthermore, we demonstrate that loss of iPLA2-VIA function leads to a number of mitochondrial abnormalities, including mitochondrial respiratory chain dysfunction, reduced ATP synthesis and abnormal mitochondrial morphology.Moreover, we show that loss of iPLA2-VIA is strongly associated with increased lipid peroxidation levels.Similar abnormalities were seen including elevated mitochondrial lipid peroxidation and mitochondrial membrane defects, as well as raised levels of cytoplasmic and mitochondrial reactive oxygen species.

View Article: PubMed Central - PubMed

Affiliation: 1 Institute of Healthy Ageing and Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, UK 2 Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK k.kinghorn@ucl.ac.uk.

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Lack of iPLA2-VIA gene activity leads to reduced survival and locomotor deficits. (A) Schematic of the Drosophila iPLA2-VIA gene, showing the position of the P-element insertion (arrowhead). The iPLA2-VIA gene has four main transcripts: iPLA2-VIA-RB, iPLA2-VIA-RC, iPLA2-VIA-RD and iPLA2-VIA-RA (FlyBase). The position of the primers has been previously shown (Malhotra et al., 2009). (B) RT-PCR analysis of iPLA2-VIA−/− flies shows that there is no mRNA expression of the iPLA2-VIA gene compared with w1118 control flies. The end products of the RT-PCR reaction were run on an agarose gel. RP49 is an internal control gene and is expressed in both the iPLA2-VIA−/− and control w1118 flies. (C) iPLA2-VIA−/− flies display an age-dependent reduction in climbing ability compared with w1118 controls (n = 45 flies per genotype, three repeats per genotype, P < 0.05). Climbing ability is expressed as a perfomance index (PI) over time in days (d). (D) Female flies lacking both iPLA2-VIA genes (iPLA2-VIA−/−) have a significantly reduced lifespan compared with w1118 control flies (P < 0.0001). Furthermore female flies lacking only one iPLA2-VIA gene (iPLA2-VIA+/−; n = 150 flies per genotype) do not display a reduction in lifespan. (E) Ubiquitous downregulation of iPLA2-VIA activity using sRNAi (UAS-iPLA2-VIA-RNAi) driven by Act5c-Gal4 (Act5c-Gal4 > UAS-iPLA2-VIA-RNAi) results in an age-dependent reduction in climbing ability compared with control UAS-iPLA2-VIA-RNAi flies (P < 0.05; n = 45 flies per genotype, three repeats each). (F) Knockdown of iPLA2-VIA activity using iPLA2-VIA sRNAi (UAS-iPLA2-VIA-RNAi) driven ubiquitously by Act5c-Gal4 (Act5c-Gal4 > UAS-iPLA2-VIA-RNAi) results in a decrease in lifespan compared with control non-expressing UAS-iPLA2-VIA-RNAi flies (P < 0.0001) or driver Act5c-Gal4 flies (P < 0.0001), similar to that seen with the  mutant flies (iPLA2-VIA−/−; n = 150 flies per genotype). (G) Knockdown of iPLA2-VIA activity using sRNAi (UAS-iPLA2-VIA-RNAi) driven in the fly nervous system using the elav-Gal4 driver results in a decrease in lifespan compared with control non-expressing UAS-iPLA2-VIA-RNAi flies (P < 0.0001), or driver elav-Gal4 (P < 0.0001; n = 150 flies per genotype), similar to that seen with the  mutant flies (iPLA2-VIA−/−).
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awv132-F1: Lack of iPLA2-VIA gene activity leads to reduced survival and locomotor deficits. (A) Schematic of the Drosophila iPLA2-VIA gene, showing the position of the P-element insertion (arrowhead). The iPLA2-VIA gene has four main transcripts: iPLA2-VIA-RB, iPLA2-VIA-RC, iPLA2-VIA-RD and iPLA2-VIA-RA (FlyBase). The position of the primers has been previously shown (Malhotra et al., 2009). (B) RT-PCR analysis of iPLA2-VIA−/− flies shows that there is no mRNA expression of the iPLA2-VIA gene compared with w1118 control flies. The end products of the RT-PCR reaction were run on an agarose gel. RP49 is an internal control gene and is expressed in both the iPLA2-VIA−/− and control w1118 flies. (C) iPLA2-VIA−/− flies display an age-dependent reduction in climbing ability compared with w1118 controls (n = 45 flies per genotype, three repeats per genotype, P < 0.05). Climbing ability is expressed as a perfomance index (PI) over time in days (d). (D) Female flies lacking both iPLA2-VIA genes (iPLA2-VIA−/−) have a significantly reduced lifespan compared with w1118 control flies (P < 0.0001). Furthermore female flies lacking only one iPLA2-VIA gene (iPLA2-VIA+/−; n = 150 flies per genotype) do not display a reduction in lifespan. (E) Ubiquitous downregulation of iPLA2-VIA activity using sRNAi (UAS-iPLA2-VIA-RNAi) driven by Act5c-Gal4 (Act5c-Gal4 > UAS-iPLA2-VIA-RNAi) results in an age-dependent reduction in climbing ability compared with control UAS-iPLA2-VIA-RNAi flies (P < 0.05; n = 45 flies per genotype, three repeats each). (F) Knockdown of iPLA2-VIA activity using iPLA2-VIA sRNAi (UAS-iPLA2-VIA-RNAi) driven ubiquitously by Act5c-Gal4 (Act5c-Gal4 > UAS-iPLA2-VIA-RNAi) results in a decrease in lifespan compared with control non-expressing UAS-iPLA2-VIA-RNAi flies (P < 0.0001) or driver Act5c-Gal4 flies (P < 0.0001), similar to that seen with the mutant flies (iPLA2-VIA−/−; n = 150 flies per genotype). (G) Knockdown of iPLA2-VIA activity using sRNAi (UAS-iPLA2-VIA-RNAi) driven in the fly nervous system using the elav-Gal4 driver results in a decrease in lifespan compared with control non-expressing UAS-iPLA2-VIA-RNAi flies (P < 0.0001), or driver elav-Gal4 (P < 0.0001; n = 150 flies per genotype), similar to that seen with the mutant flies (iPLA2-VIA−/−).

Mentions: A mutant fly stock completely lacking the Drosophila orthologue of the PLA2G6 gene, iPLA2-VIA, was obtained from the Bloomington Drosophila Stock Centre. We will refer to mutant flies lacking both iPLA2-VIA genes as iPLA2-VIA−/− henceforth. This fly strain carries a P-element insertion (EY05103) in the iPLA2-VIA gene (Fig. 1A). As previously shown, RT-PCR analysis confirmed that there was complete knockout of iPLA2-VIA gene expression in this strain with no detectable SYBR green signal produced. The end products of the RT-PCR reactions were also visualized on agarose gel electrophoresis, which confirmed the complete absence of iPLA2-VIA gene expression in the iPLA2-VIA−/− flies (Fig. 1B) (Malhotra et al., 2009). Knockout of iPLA2-VIA activity in the fly did not produce any gross changes in body size or obvious differences in morphology (data not shown). iPLA2-VIA is expressed throughout the adult fly body, including the brain and eye. The iPLA2-VIA gene displays good homology with the human PLA2G6 gene, with a 51% identity and 67% positivity (FlyBase).Figure 1


Loss of PLA2G6 leads to elevated mitochondrial lipid peroxidation and mitochondrial dysfunction.

Kinghorn KJ, Castillo-Quan JI, Bartolome F, Angelova PR, Li L, Pope S, Cochemé HM, Khan S, Asghari S, Bhatia KP, Hardy J, Abramov AY, Partridge L - Brain (2015)

Lack of iPLA2-VIA gene activity leads to reduced survival and locomotor deficits. (A) Schematic of the Drosophila iPLA2-VIA gene, showing the position of the P-element insertion (arrowhead). The iPLA2-VIA gene has four main transcripts: iPLA2-VIA-RB, iPLA2-VIA-RC, iPLA2-VIA-RD and iPLA2-VIA-RA (FlyBase). The position of the primers has been previously shown (Malhotra et al., 2009). (B) RT-PCR analysis of iPLA2-VIA−/− flies shows that there is no mRNA expression of the iPLA2-VIA gene compared with w1118 control flies. The end products of the RT-PCR reaction were run on an agarose gel. RP49 is an internal control gene and is expressed in both the iPLA2-VIA−/− and control w1118 flies. (C) iPLA2-VIA−/− flies display an age-dependent reduction in climbing ability compared with w1118 controls (n = 45 flies per genotype, three repeats per genotype, P < 0.05). Climbing ability is expressed as a perfomance index (PI) over time in days (d). (D) Female flies lacking both iPLA2-VIA genes (iPLA2-VIA−/−) have a significantly reduced lifespan compared with w1118 control flies (P < 0.0001). Furthermore female flies lacking only one iPLA2-VIA gene (iPLA2-VIA+/−; n = 150 flies per genotype) do not display a reduction in lifespan. (E) Ubiquitous downregulation of iPLA2-VIA activity using sRNAi (UAS-iPLA2-VIA-RNAi) driven by Act5c-Gal4 (Act5c-Gal4 > UAS-iPLA2-VIA-RNAi) results in an age-dependent reduction in climbing ability compared with control UAS-iPLA2-VIA-RNAi flies (P < 0.05; n = 45 flies per genotype, three repeats each). (F) Knockdown of iPLA2-VIA activity using iPLA2-VIA sRNAi (UAS-iPLA2-VIA-RNAi) driven ubiquitously by Act5c-Gal4 (Act5c-Gal4 > UAS-iPLA2-VIA-RNAi) results in a decrease in lifespan compared with control non-expressing UAS-iPLA2-VIA-RNAi flies (P < 0.0001) or driver Act5c-Gal4 flies (P < 0.0001), similar to that seen with the  mutant flies (iPLA2-VIA−/−; n = 150 flies per genotype). (G) Knockdown of iPLA2-VIA activity using sRNAi (UAS-iPLA2-VIA-RNAi) driven in the fly nervous system using the elav-Gal4 driver results in a decrease in lifespan compared with control non-expressing UAS-iPLA2-VIA-RNAi flies (P < 0.0001), or driver elav-Gal4 (P < 0.0001; n = 150 flies per genotype), similar to that seen with the  mutant flies (iPLA2-VIA−/−).
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awv132-F1: Lack of iPLA2-VIA gene activity leads to reduced survival and locomotor deficits. (A) Schematic of the Drosophila iPLA2-VIA gene, showing the position of the P-element insertion (arrowhead). The iPLA2-VIA gene has four main transcripts: iPLA2-VIA-RB, iPLA2-VIA-RC, iPLA2-VIA-RD and iPLA2-VIA-RA (FlyBase). The position of the primers has been previously shown (Malhotra et al., 2009). (B) RT-PCR analysis of iPLA2-VIA−/− flies shows that there is no mRNA expression of the iPLA2-VIA gene compared with w1118 control flies. The end products of the RT-PCR reaction were run on an agarose gel. RP49 is an internal control gene and is expressed in both the iPLA2-VIA−/− and control w1118 flies. (C) iPLA2-VIA−/− flies display an age-dependent reduction in climbing ability compared with w1118 controls (n = 45 flies per genotype, three repeats per genotype, P < 0.05). Climbing ability is expressed as a perfomance index (PI) over time in days (d). (D) Female flies lacking both iPLA2-VIA genes (iPLA2-VIA−/−) have a significantly reduced lifespan compared with w1118 control flies (P < 0.0001). Furthermore female flies lacking only one iPLA2-VIA gene (iPLA2-VIA+/−; n = 150 flies per genotype) do not display a reduction in lifespan. (E) Ubiquitous downregulation of iPLA2-VIA activity using sRNAi (UAS-iPLA2-VIA-RNAi) driven by Act5c-Gal4 (Act5c-Gal4 > UAS-iPLA2-VIA-RNAi) results in an age-dependent reduction in climbing ability compared with control UAS-iPLA2-VIA-RNAi flies (P < 0.05; n = 45 flies per genotype, three repeats each). (F) Knockdown of iPLA2-VIA activity using iPLA2-VIA sRNAi (UAS-iPLA2-VIA-RNAi) driven ubiquitously by Act5c-Gal4 (Act5c-Gal4 > UAS-iPLA2-VIA-RNAi) results in a decrease in lifespan compared with control non-expressing UAS-iPLA2-VIA-RNAi flies (P < 0.0001) or driver Act5c-Gal4 flies (P < 0.0001), similar to that seen with the mutant flies (iPLA2-VIA−/−; n = 150 flies per genotype). (G) Knockdown of iPLA2-VIA activity using sRNAi (UAS-iPLA2-VIA-RNAi) driven in the fly nervous system using the elav-Gal4 driver results in a decrease in lifespan compared with control non-expressing UAS-iPLA2-VIA-RNAi flies (P < 0.0001), or driver elav-Gal4 (P < 0.0001; n = 150 flies per genotype), similar to that seen with the mutant flies (iPLA2-VIA−/−).
Mentions: A mutant fly stock completely lacking the Drosophila orthologue of the PLA2G6 gene, iPLA2-VIA, was obtained from the Bloomington Drosophila Stock Centre. We will refer to mutant flies lacking both iPLA2-VIA genes as iPLA2-VIA−/− henceforth. This fly strain carries a P-element insertion (EY05103) in the iPLA2-VIA gene (Fig. 1A). As previously shown, RT-PCR analysis confirmed that there was complete knockout of iPLA2-VIA gene expression in this strain with no detectable SYBR green signal produced. The end products of the RT-PCR reactions were also visualized on agarose gel electrophoresis, which confirmed the complete absence of iPLA2-VIA gene expression in the iPLA2-VIA−/− flies (Fig. 1B) (Malhotra et al., 2009). Knockout of iPLA2-VIA activity in the fly did not produce any gross changes in body size or obvious differences in morphology (data not shown). iPLA2-VIA is expressed throughout the adult fly body, including the brain and eye. The iPLA2-VIA gene displays good homology with the human PLA2G6 gene, with a 51% identity and 67% positivity (FlyBase).Figure 1

Bottom Line: Furthermore, we demonstrate that loss of iPLA2-VIA function leads to a number of mitochondrial abnormalities, including mitochondrial respiratory chain dysfunction, reduced ATP synthesis and abnormal mitochondrial morphology.Moreover, we show that loss of iPLA2-VIA is strongly associated with increased lipid peroxidation levels.Similar abnormalities were seen including elevated mitochondrial lipid peroxidation and mitochondrial membrane defects, as well as raised levels of cytoplasmic and mitochondrial reactive oxygen species.

View Article: PubMed Central - PubMed

Affiliation: 1 Institute of Healthy Ageing and Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, UK 2 Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK k.kinghorn@ucl.ac.uk.

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Related in: MedlinePlus