Non-apoptotic caspase activation ensures the homeostasis of ovarian somatic stem cells.

Current evidence has associated caspase activation with the regulation of basic cellular functions without causing apoptosis. Malfunction of non-apoptotic caspase activities may contribute to specific neurological disorders, metabolic diseases, autoimmune conditions and cancers. However, our understanding of non-apoptotic caspase functions remains limited. Here, we show that non-apoptotic caspase activation prevents the intracellular accumulation of the Patched receptor in autophagosomes and the subsequent Patched-dependent induction of autophagy in Drosophila follicular stem cells. These events ultimately sustain Hedgehog signalling and the physiological properties of ovarian somatic stem cells and their progeny under moderate thermal stress. Importantly, our key findings are partially conserved in ovarian somatic cells of human origin. These observations attribute to caspases a pro-survival role under certain cellular conditions.

A LexAop > UAS > QUAS trimeric plasmid to generate inducible and interconvertible Drosophila overexpression transgenes.

The existence of three independent binary systems for conditional gene expression (Gal4/UAS; LexA/LexAop; QF/QUAS) has greatly expanded versatile genetic analyses in the Drosophila melanogaster; however, the experimental application of these tools is limited by the need to generate multiple collections of noninterchangeable transgenic fly strains for each inducible gene expression system. To address this practical limitation, we developed a modular vector that contains the regulatory elements from all three binary systems, enabling Gal4-, LexA- or QF-dependent expression of transgenes. Our methods also incorporate DNA elements that facilitate independent site-specific recombination and elimination of regulatory UAS, LexAop or QUAS modules with spatial and temporal control, thus offering unprecedented possibilities and logistical advantages for in vivo genetic modulation and efficient interconversion of overexpression transgenic fly lines.

Non-apoptotic Caspase regulation of stem cell properties.

The evolutionarily conserved family of proteins called caspases are the main factors mediating the orchestrated programme of cell suicide known as apoptosis. Since this protein family was associated with this essential biological function, the majority of scientific efforts were focused towards understanding their molecular activation and function during cell death. However, an emerging body of evidence has highlighted a repertoire of non-lethal roles within a large variety of cell types, including stem cells. Here we intend to provide a comprehensive overview of the key role of caspases as regulators of stem cell properties. Finally, we briefly discuss the possible pathological consequences of caspase malfunction in stem cells, and the therapeutic potential of caspase regulation applied to this context.

An AMPK-dependent regulatory pathway in tau-mediated toxicity.

Neurodegenerative tauopathies are characterised by accumulation of hyperphosphorylated tau aggregates primarily degraded by autophagy. The 5'AMP-activated protein kinase (AMPK) is expressed in most cells, including neurons. Alongside its metabolic functions, it is also known to be activated in Alzheimer's brains, phosphorylate tau, and be a critical autophagy activator. Whether it plays a neurotoxic or neuroprotective role remains unclear. In tauopathies stress conditions can result in AMPK activation, enhancing tau-mediated toxicity. Paradoxically, in these cases AMPK activation does not always lead to protective autophagic responses. Using a Drosophila in vivo quantitative approach, we have analysed the impact of AMPK and autophagy on tau-mediated toxicity, recapitulating the AMPK-mediated tauopathy condition: increased tau phosphorylation, without corresponding autophagy activation. We have demonstrated that AMPK binding to and phosphorylating tau at Ser-262, a site reported to facilitate soluble tau accumulation, affects its degradation. This phosphorylation results in exacerbation of tau toxicity and is ameliorated via rapamycin-induced autophagy stimulation. Our findings support the development of combinatorial therapies effective at reducing tau toxicity targeting tau phosphorylation and AMPK-independent autophagic induction. The proposed in vivo tool represents an ideal readout to perform preliminary screening for drugs promoting this process.

Correlation of NM23-H1 cytoplasmic expression with metastatic stage in human prostate cancer tissue.

Nm23-H1 has been identified as a metastatic suppressor gene in murine melanoma cell lines. Several functions have been attributed to its activity in cancer, including a histidine kinase activity, DNA repair, and regulation of other proteins involved in metastatic formation. While in breast cancer, NM23-H1 overexpression indicates a benign status through impairing progression of disease, its function is opposite in other cancers; e.g., neuroblastoma. To further understand this dichotomy of function in cancer, we have analyzed its function in prostate cancer, in which the relationship between NM23-H1 expression and prognostic state is today controversial. In vitro, overexpression of NM23-H1 in PC3 cells inhibited their cell motility, while downregulation of NM23-H1 expression in these cells by RNA interference showed enhanced cell motility. Immunohistochemistry analysis performed on 346 prostate cancer tissue samples showed a relationship between high levels of NM23-H1 expression in the nuclei of these tumorigenic cells and elevated Gleason score, with high levels of NM23-H1 cytoplasmic staining related to metastatic stage. This retrospective survival study demonstrates that high levels of NM23-H1 expression in the cytoplasm determine recurrence of prostate-specific antigen levels only in those patients with metastatic disease. Our findings suggest a correlation between high levels of NM23-H1 protein in the cytoplasm of the cells and progression of prostate cancer to metastasis, thus definitively identifying NM23-H1 as a new negative prognostic marker in prostate cancer.

Diminution of eIF4E activity suppresses parkin mutant phenotypes.

Mutations in the human parkin (PARK2) gene cause autosomal recessive-juvenile Parkinson's disease (AR-JP). In Drosophila melanogaster, mutant parkin alleles display a broad range of phenotypic alterations, including female infertility. Here we report that reducing the level of eukaryotic translation initiation factor 4E (eIF4E) activity specifically rescues the female sterile phenotypes associated with the parkin(P23) mutant allele. Additional defects, including reduction of pupal viability and body size, are also entirely recovered in both male and female flies of the abovementioned genotype. We further show that a null eIF4E-binding protein (4E-BP) allele counteracts the in vivo effects produced, in a parkin(P23) mutant background, by the reduction of functional eIF4E copy number. Moreover, Parkin and eIF4E interact in vitro and co-localize at the posterior end of developing oocytes. Finally, we show that eIF4E is over-expressed in parkin(P23) mutant ovaries as compared to wild-types. Taken together, our data are consistent with the idea that Parkin and eIF4E act in a common pathway, likely modulating cap-dependent translation initiation events.

The Nm23-H1-h-Prune complex in cellular physiology: a 'tip of the iceberg' protein network perspective.

Nm23-H1 (also known as NDPKA) and h-Prune form a protein complex that is part of a little-understood protein network. Modifications of this complex correlate with cancer status. Here, we focus on the role of the Nm23-H1-h-Prune complex in cellular physiology, through an analysis of the balance between the 'bound' and 'non-bound' states of Nm23-H1 and h-Prune, whereby we speculate on the 'read-out' during cell homeostasis under non-balanced conditions. We have analysed the biochemical activities of both Nm23-H1 and h-Prune alone and in combination, focussing on the anti-metastatic activity of Nm23-H1. We have then investigated the cellular mechanisms responsible for the formation of the Nm23-H1-h-Prune complex. To evaluate the importance of the equilibrium between the formation of the Nm23-H1-h-Prune complex and the 'free' levels of Nm23-H1 and h-Prune, we propose a model based on a pro-cancer condition where this equilibrium is negatively affected.

The translational repressor Cup associates with the adaptor protein Miranda and the mRNA carrier Staufen at multiple time-points during Drosophila oogenesis.

In Drosophila melanogaster, Cup acts as a translational regulator during oocyte maturation and early embryogenesis. In this report, we show that Cup associates with Miranda, an adaptor protein involved in localization of specific mRNA complexes in both neuroblasts and oocytes. miranda and cup also interact genetically, since reducing miranda activity worsens the oogenesis defects associated with different cup mutant alleles. miranda mRNA is first detected within the cytoplasm of egg chambers during early oogenesis, coincidentally with very low levels of Miranda protein. We furthermore show that Cup interacts with Staufen, a protein involved in mRNA localization during oogenesis and nervous system development, and the two proteins co-localize within the posterior cytoplasm of late oocytes. Our results substantiate the idea that Cup is a multi-functional protein cooperating with different protein partners to direct egg chamber development at multiple time-points.

dSTAM expression pattern during wild type and mutant egg chamber development in D. melanogaster.

STAM (signal-transducing adaptor molecule) is a protein highly conserved from yeast to mammals. In Drosophila melanogaster the basic molecular architecture of the protein is comprised of a N-terminal VHS domain, an ubiquitin-interacting motif and a central Src homology-3 domain. In this paper we examine the expression pattern of the stam gene and the localisation of the STAM protein during D. melanogaster oogenesis. Its transcript is present throughout egg chamber development in all germ-line cells, including the oocyte. dSTAM is firstly detected in germarial region 2, where the protein is present in the newly formed germ-line cysts and is mainly accumulated into the oocyte. As oogenesis proceeds, dSTAM is enriched in the perinuclear region of the nurse cells and is also found in the somatic polar follicular cells. In the oocyte, the protein is more abundant posteriorly and becomes restricted to the posterior pole just before disappearing at stage 10b. We show that dSTAM localisation is unaffected in the oocyte of grk mutant egg chambers, indicating that it is not dependent on the polarity of the microtubule network. In contrast, dSTAM distribution is remarkably altered in cup mutant oocytes where the protein accumulates in a round central spot and never reaches the posterior pole.