PDIA6 and Maspin in Prostate Cancer.

BACKGROUND/AIM: PDIA6 is a disulphide isomerase of the PDI family, known to mediate disulphide bond formation in the endoplasmic reticulum. However, PDI-related proteins also function in other parts of the cell and PDIA6 has been shown to be involved in many types of cancers. We previously identified PDIA6 as a putative Maspin interactor. Maspin has itself been implicated in prostate cancer progression. Our aim was to further explore the roles of Maspin in prostate cancer and establish whether PDIA6 is also involved in prostate cancer. MATERIALS AND METHODS: RNA levels of PDIA6 and Maspin in prostate cell lines were measured using RT-PCR. Bioinformatics analysis of the TCGA database was used to find RNA levels of PDIA6 and Maspin in prostate cancer. siRNAs were used to knock-down PDIA6, and proliferation and migration assays were conducted on those cells. RESULTS: PDIA6 and Maspin RNA were shown to be expressed at varying levels in prostate cell lines. RNAseq data showed that PDIA6 expression was significantly increased in prostate adenocarcinoma samples, while Maspin RNA expression was decreased. When PDIA6 expression was knocked-down using siRNA in prostate cell lines, proliferation was decreased substantially in the two prostate cancer cell lines (DU145 and PC3) and also decreased in the normal prostate cell line (PNT1a), though less strongly. CONCLUSION: PDIA6 expression is higher in prostate cancer cells compared to normal prostate cells. Decreasing PDIA6 expression decreases proliferation. Thus, PDIA6 is a promising target for prostate cancer therapeutics.

Mitochondrial electron transport chain defects modify Parkinson's disease phenotypes in a Drosophila model.

INTRODUCTION: Mitochondrial defects have been implicated in Parkinson's disease (PD) since complex I poisons were found to cause accelerated parkinsonism in young people in the early 1980s. More evidence of mitochondrial involvement arose when many of the genes whose mutations caused inherited PD were discovered to be subcellularly localized to mitochondria or have mitochondrial functions. However, the details of how mitochondrial dysfunction might impact or cause PD remain unclear. The aim of our study was to better understand mitochondrial dysfunction in PD by evaluating mitochondrial respiratory complex mutations in a Drosophila melanogaster (fruit fly) model of PD. METHODS: We have conducted a targeted heterozygous enhancer/suppressor screen using Drosophila mutations within mitochondrial electron transport chain (ETC) genes against a null PD mutation in parkin. The interactions were assessed by climbing assays at 2-5 days as an indicator of motor function. A strong enhancer mutation in COX5A was examined further for L-dopa rescue, oxygen consumption, mitochondrial content, and reactive oxygen species. A later timepoint of 16-20 days was also investigated for both COX5A and a suppressor mutation in cyclope. Generalized Linear Models and similar statistical tests were used to verify significance of the findings. RESULTS: We have discovered that mutations in individual genes for subunits within the mitochondrial respiratory complexes have interactions with parkin, while others do not, irrespective of complex. One intriguing mutation in a complex IV subunit (cyclope) shows a suppressor rescue effect at early time points, improving the gross motor defects caused by the PD mutation, providing a strong candidate for drug discovery. Most mutations, however, show varying degrees of enhancement or slight suppression of the PD phenotypes. Thus, individual mitochondrial mutations within different oxidative phosphorylation complexes have different interactions with PD with regard to degree and direction. Upon further investigation of the strongest enhancer (COX5A), the mechanism by which these interactions occur initially does not appear to be based on defects in ATP production, but rather may be related to increased levels of reactive oxygen species. CONCLUSIONS: Our work highlights some key subunits potentially involved in mechanisms underlying PD pathogenesis, implicating ETC complexes other than complex I in PD.

A Drosophila chromatin factor interacts with the Piwi-interacting RNA mechanism in niche cells to regulate germline stem cell self-renewal.

Stem cell research has been focused on niche signaling and epigenetic programming of stem cells. However, epigenetic programming of niche cells remains unexplored. We showed previously that Piwi plays a crucial role in Piwi-interacting RNA-mediated epigenetic regulation and functions in the niche cells to maintain germline stem cells (GSCs) in the Drosophila ovary. To investigate the epigenetic programming of niche cells by Piwi, we screened mutations in the Polycomb and trithorax group genes, and an enhancer of Polycomb and trithorax called corto, for their potential genetic interaction with piwi. corto encodes a chromatin protein. corto mutations restored GSC division in mutants of piwi and fs(1)Yb (Yb), a gene that regulates piwi expression in niche cells to maintain GSCs. Consistent with this, corto appears to be expressed in the niche cells and is not required in the germline. Furthermore, in corto-suppressed Yb mutants, the expression of hedgehog (hh) is restored in niche cells, which is likely responsible for corto suppression of the GSC and somatic stem cell defects of Yb mutants. These results reveal a novel epigenetic mechanism involving Corto and Piwi that defines the fate and signaling function of niche cells in maintaining GSCs.

Mitochondrial DNA mutations affect calcium handling in differentiated neurons.

Mutations in the mitochondrial genome are associated with a wide range of neurological symptoms, but many aspects of the basic neuronal pathology are not understood. One candidate mechanism, given the well-established role of mitochondria in calcium buffering, is a deficit in neuronal calcium homoeostasis. We therefore examined calcium responses in the neurons derived from various 'cybrid' embryonic stem cell lines carrying different mitochondrial DNA mutations. Brief ( approximately 50 ms), focal glutamatergic stimuli induced a transient rise in intracellular calcium concentration, which was visualized by bulk loading the cells with the calcium dye, Oregon Green BAPTA-1. Calcium entered the neurons through N-methyl-d-aspartic acid and voltage-gated calcium channels, as has been described in many other neuronal classes. Intriguingly, while mitochondrial mutations did not affect the calcium transient in response to single glutamatergic stimuli, they did alter the responses to repeated stimuli, with each successive calcium transient decaying ever more slowly in mitochondrial mutant cell lines. A train of stimuli thus caused intracellular calcium in these cells to be significantly elevated for many tens of seconds. These results suggest that calcium-handling deficits are likely to contribute to the pathological phenotype seen in patients with mitochondrial DNA mutations.

Mechanism of neurodegeneration of neurons with mitochondrial DNA mutations.

Mutations of mitochondrial DNA are associated with a wide spectrum of disorders, primarily affecting the central nervous system and muscle function. The specific consequences of mitochondrial DNA mutations for neuronal pathophysiology are not understood. In order to explore the impact of mitochondrial mutations on neuronal biochemistry and physiology, we have used fluorescence imaging techniques to examine changes in mitochondrial function in neurons differentiated from mouse embryonic stem-cell cybrids containing mitochondrial DNA polymorphic variants or mutations. Surprisingly, in neurons carrying a severe mutation in respiratory complex I (<10% residual complex I activity) the mitochondrial membrane potential was significantly increased, but collapsed in response to oligomycin, suggesting that the mitochondrial membrane potential was maintained by the F(1)F(o) ATPase operating in 'reverse' mode. In cells with a mutation in complex IV causing approximately 40% residual complex IV activity, the mitochondrial membrane potential was not significantly different from controls. The rate of generation of mitochondrial reactive oxygen species, measured using hydroethidium and signals from the mitochondrially targeted hydroethidine, was increased in neurons with both the complex I and complex IV mutations. Glutathione was depleted, suggesting significant oxidative stress in neurons with a complex I deficiency, but not in those with a complex IV defect. In the neurons with complex I deficiency but not the complex IV defect, neuronal death was increased and was attenuated by reactive oxygen species scavengers. Thus, in neurons with a severe mutation of complex I, the maintenance of a high potential by F(1)F(o) ATPase activity combined with an impaired respiratory chain causes oxidative stress which promotes cell death.

Screens for piwi suppressors in Drosophila identify dosage-dependent regulators of germline stem cell division.

The Drosophila piwi gene is the founding member of the only known family of genes whose function in stem cell maintenance is highly conserved in both animal and plant kingdoms. piwi mutants fail to maintain germline stem cells in both male and female gonads. The identification of piwi-interacting genes is essential for understanding how stem cell divisions are regulated by piwi-mediated mechanisms. To search for such genes, we screened the Drosophila third chromosome ( approximately 36% of the euchromatic genome) for suppressor mutations of piwi2 and identified six strong and three weak piwi suppressor genes/sequences. These genes/sequences interact negatively with piwi in a dosage-sensitive manner. Two of the strong suppressors represent known genes--serendipity-delta and similar, both encoding transcription factors. These findings reveal that the genetic regulation of germline stem cell division involves dosage-sensitive mechanisms and that such mechanisms exist at the transcriptional level. In addition, we identified three other types of piwi interactors. The first type consists of deficiencies that dominantly interact with piwi2 to cause male sterility, implying that dosage-sensitive regulation also exists in the male germline. The other two types are deficiencies that cause lethality and female-specific lethality in a piwi2 mutant background, revealing the zygotic function of piwi in somatic development.