The protective roles of phosphorylated heat shock protein 27 in human cells harboring myoclonus epilepsy with ragged-red fibers A8344G mtDNA mutation.

Mitochondrial DNA (mtDNA) mutations are associated with a large number of neuromuscular diseases. Myoclonus epilepsy with ragged-red fibers (MERRF) syndrome is a mitochondrial disease inherited through the maternal lineage. The most common mutation in MERRF syndrome, the A8344G mutation of mtDNA, is associated with severe defects in mitochondrial protein synthesis, which impair the assembly and function of the respiratory chain. We have previously shown that there is a decreased level of heat shock protein 27 (HSP27) in lymphoblastoid cells derived from a MERRF patient and in cytoplasmic hybrids (cybrids) harboring the A8344G mutation of mtDNA. In the present study, we found a dramatic decrease in the level of phosphorylated HSP27 (p-HSP27) in the mutant cybrids. Even though the steady-state level of p-HSP27 was reduced in the mutant cybrids, normal phosphorylation and dephosphorylation were observed upon exposure to stress, indicating normal kinase and phosphatase activities. To explore the roles that p-HSP27 may play, transfection experiments with HSP27 mutants, in which three specific serines were replaced with alanine or aspartic acid, showed that the phosphomimicking HSP27 desensitized mutant cybrids to apoptotic stress induced by staurosporine (STS). After heat shock stress, p-HSP27 was found to enter the nucleus immediately, and with a prolonged interval of recovery, p-HSP27 returned to the cytoplasm in wild-type cybrids but not in mutant cybrids. The translocation of p-HSP27 was correlated with cell viability, as shown by the increased number of apoptotic cells after p-HSP27 returned to the cytoplasm. In summary, our results demonstrate that p-HSP27 provides significant protection when cells are exposed to different stresses in the cell model of MERRF syndrome. Therapeutic agents targeting anomalous HSP27 phosphorylation might represent a potential treatment for mitochondrial diseases.

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.