Age‐Related Effects on the Biological Clock and its Behavioral Output in a Primate

In humans, activity rhythms become fragmented and attenuated in the elderly. This suggests an alteration of the circadian system per se that could in turn affect the expression of biological rhythms. In primates, very few studies have analyzed the effect of aging on the circadian system. The mouse lemur provides a unique model of aging in non‐human primates. To assess the effect of aging on the circadian system of this primate, we recorded the circadian and daily rhythms of locomotor activity of mouse lemurs of various ages. We also examined age‐related changes in the daily rhythm of immunoreactivities for vasoactive intestinal polypeptide (VIP) and arginine‐vasopressin (AVP) in suprachiasmatic nucleus neurons (SCN), two major peptides of the biological clock. Compared to adult animals, aged mouse lemurs showed a significant increase in daytime activity and an advanced activity onset. Moreover, when maintained in constant dim red light, aged animals exhibited a shortening of the free‐running period compared to adult animals. In adults, AVP immunoreactivity (ir) peaked during the second part of the day, and VIP ir peaked during the night. In aged mouse lemurs, the peaks of AVP ir and VIP ir were significantly shifted with no change in amplitude. AVP ir was most intense at the beginning of the night; whereas, VIP ir peaked at the beginning of the daytime. A weakened oscillator could account for the rhythmic disorders often observed in the elderly. Changes in the daily rhythms of AVP ir and VIP ir may affect the ability of the SCN to transmit rhythmic information to other neural target sites, and thereby modify the expression of some biological rhythms.     

Unique gene alterations are induced in FACS‐purified Fos‐positive neurons activated during cue‐induced relapse to heroin seeking

Cue‐induced heroin seeking after prolonged withdrawal is associated with neuronal activation and altered gene expression in prefrontal cortex (PFC). However, these previous studies assessed gene expression in all neurons regardless of their activity state during heroin seeking. Using Fos as a marker of neural activity, we describe distinct molecular alterations induced in activated versus non‐activated neurons during cue‐induced heroin seeking after prolonged withdrawal. We trained rats to self‐administer heroin for 10 days (6 h/day) and assessed cue‐induced heroin seeking in extinction tests after 14 or 30 days. We used fluorescent‐activated cell sorting (FACS) to purify Fos‐positive and Fos‐negative neurons from PFC 90 min after extinction testing. Flow cytometry showed that Fos‐immunoreactivity was increased in less than 10% of sparsely distributed PFC neurons. mRNA levels of the immediate early genes fosB, arc, egr1, and egr2, as well as npy and map2k6, were increased in Fos‐positive, but not Fos‐negative, neurons. In support of these findings, double‐label immunohistochemistry indicated substantial coexpression of neuropeptide Y (NPY)‐ and Arc‐immunoreactivity in Fos‐positive neurons. Our data indicate that cue‐induced relapse to heroin seeking after prolonged withdrawal induces unique molecular alterations within activated PFC neurons that are distinct from those observed in the surrounding majority of non‐activated neurons.     

Species Diversity,Phylogeny and Large Scale Biogeographic Patterns of the Genus Padina (Phaeophyceae,Dictyotales)

The brown algal genus Padina (Dictyotales, Phaeophyceae) is distributed worldwide in tropical and temperate seas. Global species diversity and distribution ranges, however, remain largely unknown. Species‐level diversity was reassessed using DNA‐based, algorithmic species delineation techniques based on cox3 and rbcL sequence data from 221 specimens collected worldwide. This resulted in estimates ranging from 39 to 61 putative species (ESUs), depending on the technique as well as the locus. We discuss the merits, potential pitfalls, and evolutionary and biogeographic significance of algorithmic species delineation. We unveil patterns whereby ESUs are in all but one case restricted to either the Atlantic or Indo‐Pacific Ocean. Within ocean basins we find evidence for the vast majority of ESUs to be confined to a single marine realm. Exceptions, whereby ESUs span up to three realms, are located in the Indo‐Pacific Ocean. Patterns of range‐restricted species likely arise by repeated founder events and subsequent peripatric speciation, hypothesized to dominate speciation mechanisms for coastal marine organisms in the Indo‐Pacific. Using a three‐gene (cox3, psaA and rbcL), relaxed molecular clock phylogenetic analysis we estimated divergence times, providing a historical framework to interpret biogeographic patterns.     

Soluble Melanoma Cell Adhesion Molecule (sMCAM/sCD146) Promotes Angiogenic Effects on Endothelial Progenitor Cells through Angiomotin

The melanoma cell adhesion molecule (CD146) contains a circulating proteolytic variant (sCD146), which is involved in inflammation and angiogenesis. Its circulating level is modulated in different pathologies, but its intracellular transduction pathways are still largely unknown. Using peptide pulldown and mass spectrometry, we identified angiomotin as a sCD146-associated protein in endothelial progenitor cells (EPC). Interaction between angiomotin and sCD146 was confirmed by enzyme-linked immunosorbent assay (ELISA), homogeneous time-resolved fluorescence, and binding of sCD146 on both immobilized recombinant angiomotin and angiomotin-transfected cells. Silencing angiomotin in EPC inhibited sCD146 angiogenic effects, i.e. EPC migration, proliferation, and capacity to form capillary-like structures in Matrigel. In addition, sCD146 effects were inhibited by the angiomotin inhibitor angiostatin and competition with recombinant angiomotin. Finally, binding of sCD146 on angiomotin triggered the activation of several transduction pathways that were identified by antibody array. These results delineate a novel signaling pathway where sCD146 binds to angiomotin to stimulate a proangiogenic response. This result is important to find novel target cells of sCD146 and for the development of therapeutic strategies based on EPC in the treatment of ischemic diseases.     

Inhibition of melanoma development in the Nras(Q61K)::Ink4a−/− mouse model by the small molecule BI‐69A11

To date, there are no effective therapies for tumors bearing NRAS mutations, which are present in 15–20% of human melanomas. Here we extend our earlier studies where we demonstrated that the small molecule BI‐69A11 inhibits the growth of melanoma cell lines. Gene expression analysis revealed the induction of interferon‐ and cell death‐related genes that were associated with responsiveness of melanoma cell lines to BI‐69A11. Strikingly, the administration of BI‐69A11 inhibited melanoma development in genetically modified mice bearing an inducible form of activated Nras and a deletion of the Ink4a gene (Nras^(Q61K)::Ink4a^?/?). Biweekly administration of BI‐69A11 starting at 10 weeks or as late as 24 weeks after the induction of mutant Nras expression inhibited melanoma development (100 and 36%, respectively). BI‐69A11 treatment did not inhibit the development of histiocytic sarcomas, which constitute about 50% of the tumors in this model. BI‐69A11‐resistant Nras^(Q61K)::Ink4a^?/? tumors exhibited increased CD45 expression, reflective of immune cell infiltration and upregulation of gene networks associated with the cytoskeleton, DNA damage response, and small molecule transport. The ability to attenuate the development of NRAS mutant melanomas supports further development of BI‐69A11 for clinical assessment.     

A Huntingtin Peptide Inhibits PolyQ-Huntingtin Associated Defects

Background

Huntington’s disease (HD) is caused by the abnormal expansion of the polyglutamine tract in the human Huntingtin protein (polyQ-hHtt). Although this mutation behaves dominantly, huntingtin loss of function also contributes to HD pathogenesis. Indeed, wild-type Huntingtin plays a protective role with respect to polyQ-hHtt induced defects.

Methodology/Principal Findings

The question that we addressed here is what part of the wild-type Huntingtin is responsible for these protective properties. We first screened peptides from the Huntingtin protein in HeLa cells and identified a 23 aa peptide (P42) that inhibits polyQ-hHtt aggregation. P42 is part of the endogenous Huntingtin protein and lies within a region rich in proteolytic sites that plays a critical role in the pathogenesis process. Using a Drosophila model of HD, we tested the protective properties of this peptide on aggregation, as well as on different polyQ-hHtt induced neuronal phenotypes: eye degeneration (an indicator of cell death), impairment of vesicular axonal trafficking, and physiological behaviors such as larval locomotion and adult survival. Together, our results demonstrate high protective properties for P42 in vivo, in whole animals. These data also demonstrate a specific role of P42 on Huntington’s disease model, since it has no effect on other models of polyQ-induced diseases, such as spinocerebellar ataxias.

Conclusions/Significance

Altogether our data show that P42, a 23 aa-long hHtt peptide, plays a protective role with respect to polyQ-hHtt aggregation as well as cellular and behavioral dysfunctions induced by polyQ-hHtt in vivo. Our study also confirms the correlation between polyQ-hHtt aggregation and neuronal defects. Finally, these results strongly suggest a therapeutic potential for P42, specific of Huntington’s disease.     

Endothelial Protein C Receptor Function in Murine and Human Breast Cancer Development

Several markers identify cancer stem cell-like populations, but little is known about the functional roles of stem cell surface receptors in tumor progression. Here, we show that the endothelial protein C receptor (EPCR), a stem cell marker in hematopoietic, neuronal and epithelial cells, is crucial for breast cancer growth in the orthotopic microenvironment of the mammary gland. Mice with a hypomorphic allele of EPCR show reduced tumor growth in the PyMT-model of spontaneous breast cancer development and deletion of EPCR in established PyMT tumor cells significantly attenuates transplanted tumor take and growth. We find expansion of EPCR⁺ cancer stem cell-like populations in aggressive, mammary fat pad-enhanced human triple negative breast cancer cells. In this model, EPCR-expressing cells have markedly increased mammosphere- and tumor-cell initiating activity compared to another stable progenitor-like subpopulation present at comparable frequency. We show that receptor blocking antibodies to EPCR specifically attenuate in vivo tumor growth initiated by either EPCR⁺ cells or the heterogenous mixture of EPCR⁺ and EPCR^- cells. Furthermore, we have identified tumor associated macrophages as a major source for recognized ligands of EPCR, suggesting a novel mechanism by which cancer stem cell-like populations are regulated by innate immune cells in the tumor microenvironment.