Effects of carbonic anhydrase-related protein VIII on human cells harbouring an A8344G mitochondrial DNA mutation.

MERRF (myoclonus epilepsy associated with ragged-red fibres) is a maternally inherited mitochondrial encephalomyopathy with various syndromes involving both muscular and nervous systems. The most common mutation in MERRF syndrome, the A8344G mutation in mtDNA, has been associated with severe defects in the respiratory function of mitochondria. In the present study, we show that there is a significant decrease in CA8 (carbonic anhydrase-related protein VIII) in cybrids harbouring the MERRF A8344G mutation. CA8 deficiency and mutations were found to be associated with a distinctive lifelong gait disorder in wdl (Waddles) mice and novel syndromes characterized by cerebellar ataxia and mental retardation in humans. The results of the present study showed that overexpression of CA8 in MERRF cybrids significantly decreased cell death induced by STS (staurosporine) treatment, suggesting a protective function of CA8 in cells harbouring the A8344G mutation of mtDNA. Interestingly, an increase in the formation of LC3-II (microtubule-associated protein 1 light chain 3-II) was found in the cybrids with down-regulated CA8 expression, suggesting that reduced expression of CA8 leads to autophagy activation. Furthermore, cybrids exhibiting down-regulated CA8 showed increased cytosolic Ca2+ signals and reduced levels of phospho-Akt compared with those in the cybrids with overexpressed CA8, indicating that phospho-Akt is involved in the protection of cells by CA8. Our findings suggest that CA8 is involved in the autophagic pathway and may have a protective role in cultured cells from patients with MERRF. Targeting CA8 and the downstream autophagic pathway might help develop therapeutic agents for treatment of MERRF syndrome in the future.

Decreased heat shock protein 27 expression and altered autophagy in human cells harboring A8344G mitochondrial DNA mutation.

Mitochondrial DNA (mtDNA) mutations are responsible for human neuromuscular diseases caused by mitochondrial dysfunction. Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a maternally inherited mitochondrial encephalomyopathy with various syndromes involving both muscular and nervous systems. The most common mutation in MERRF syndrome, A8344G mutation in mtDNA, has been associated with severe defects in protein synthesis. This defect impairs assembly of complexes in electron transport chain and results in decreased respiratory function of mitochondria. In this study, we showed a significant decrease of the heat shock protein 27 (Hsp27) in lymphoblastoid cells derived from a MERRF patient and in cybrid cells harboring MERRF A8344G mutation. However, normal cytoplasmic distributions of Hsp27 and normal heat shock responses were observed in both wild type and mutant cybrids. Furthermore, overexpression of wild type Hsp27 in mutant MERRF cybrids significantly decreased cell death under staurosporine (STS) treatment, suggesting a protective function of Hsp27 in cells harboring the A8344G mutation of mtDNA. Meanwhile, reverse transcriptase PCR showed no difference in the mRNA level between normal and mutant cybrids, indicating that alterations may occur at the protein level. Evidenced by the decreased levels of Hsp27 upon treatment with proteasome inhibitor, starvation and rapamycin and the accumulation of Hsp27 upon lysosomal inhibitor treatment; Hsp27 may be degraded by the autophagic pathway. In addition, the increased formation of LC3-II and autophagosomes was found in MERRF cybrids under the basal condition, indicating a constitutively-activated autophagic pathway. It may explain, at least partially, the faster turnover of Hsp27 in MERRF cybrids. This study provides information for us to understand that Hsp27 is degraded through the autophagic pathway and that Hsp27 may have a protective role in MERRF cells. Regulating Hsp27 and the autophagic pathway might help develop therapeutic solutions for treatment of MERRF syndrome in the future.