Stress amplifies sex differences in primate prefrontal profiles of gene expression

Background

Stress is a recognized risk factor for mood and anxiety disorders that occur more often in women than men. Prefrontal brain regions mediate stress coping, cognitive control, and emotion. Here, we investigate sex differences and stress effects on prefrontal cortical profiles of gene expression in squirrel monkey adults.

Methods

Dorsolateral, ventrolateral, and ventromedial prefrontal cortical regions from 18 females and 12 males were collected after stress or no-stress treatment conditions. Gene expression profiles were acquired using HumanHT-12v4.0 Expression BeadChip arrays adapted for squirrel monkeys.

Results

Extensive variation between prefrontal cortical regions was discerned in the expression of numerous autosomal and sex chromosome genes. Robust sex differences were also identified across prefrontal cortical regions in the expression of mostly autosomal genes. Genes with increased expression in females compared to males were overrepresented in mitogen-activated protein kinase and neurotrophin signaling pathways. Many fewer genes with increased expression in males compared to females were discerned, and no molecular pathways were identified. Effect sizes for sex differences were greater in stress compared to no-stress conditions for ventromedial and ventrolateral prefrontal cortical regions but not dorsolateral prefrontal cortex.

Conclusions

Stress amplifies sex differences in gene expression profiles for prefrontal cortical regions involved in stress coping and emotion regulation. Results suggest molecular targets for new treatments of stress disorders in human mental health.

     

Cell Proliferation and Epidermal Growth Factor Signaling in Non-small Cell Lung Adenocarcinoma Cell Lines Are Dependent on Rin1

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Δ) complemented the Rin1 depletion effects, and overexpression of Rin1Δ had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature.Internalization of epidermal growth factor receptors (EGFR)² and their subsequent delivery to lysosomes play key roles in attenuating EGF-mediated signaling cascades (1, 2). The proper delivery of EGFR into lysosomes for degradation requires a series of highly regulated targeting and delivery events. Following ligand binding, EGFR is internalized via endocytic vesicles that are subsequently targeted to early endosomes. This targeting event is mediated by the small GTPase, Rab5 (3, 4). Once delivered to the early endosome, receptors that are destined for degradation are incorporated into vesicles that bud into the lumen of the endosome, forming the multivesicular body (reviewed in Refs. 5, 6). Sequestration of the activated cytoplasmic domain of EGFR into the intralumenal vesicles of the multivesicular body effectively terminates receptor signaling (7). Subsequent fusion of the multivesicular body with lysosomes delivers the intralumenal vesicles and their contents into the lumen of the lysosome where they are degraded (reviewed in Refs. 8–10). Inactivating mutations in Rab5 disrupt the delivery of cell surface receptors, such as EGFR, to early endosomes, thereby inhibiting receptor trafficking to the lysosome and receptor degradation (11, 12). Therefore, activation of Rab5 is a key point of regulation for EGFR signaling.Rab5 cycles between an inactive GDP-bound state and an active GTP-bound state, and Rab5 activation requires the exchange of GDP to GTP. This exchange is catalyzed by guanine nucleotide exchange factors (GEFs) that are specific to the Rab5 family of proteins (reviewed in Ref. 13). Rab5 family GEFs all contain a catalytic vacuolar protein sorting 9 (Vps9) domain that facilitates the GDP to GTP exchange (14–17). Many Rab5 GEFs contain other functional domains that are involved in cell signaling events (13). Rin1 is a good example of a multidomain Rab5 GEF. In addition to the Vps9 domain, Rin1 also contains an Src homology 2 domain, a proline-rich domain, and a Ras association domain. Rin1 was originally identified through its ability to interact with active Ras (18), and a role for Rin1 in a number of cell signaling systems has been established, including EGF-mediated signaling (19–21). Rin1 directly interacts with the activated EGFR through its Src homology 2 domain (22). Furthermore, Ras occupation of the Rin1 Ras association domain positively impacts the Rab5 GEF activity of Rin1, which promotes EGFR internalization and attenuation in fibroblasts (23). However, Rin1 expression is up-regulated in several types of cancers, including squamous cell carcinoma (24), colorectal cancer (25), and cervical cancer (26), through duplications or rearrangements of the RIN1 locus. These studies suggest that Rin1 may also play a role in enhancing cell proliferation.It is well established that a large percentage of non-small cell lung adenocarcinomas exhibit up-regulation of EGFR and aberrant signaling through the Ras/MAPK pathway (reviewed in Ref. 27). In addition, a recent study examining 188 human lung adenocarcinomas identified that 132 of 188 tumor samples exhibited mutations relating to the Ras/MAPK signaling pathway (28). Accordingly, the role of Rin1 in non-small cell lung adenocarcinoma was addressed. Examination of a panel of non-small cell lung adenocarcinoma lines (including A549) revealed enhanced Rin1 expression relative to a nontransformed lung epithelial cell line (BEAS-2B). Depletion of Rin1 from A549 cells resulted in decreased proliferation. This decrease correlated with a reduction in EGF-activated ERK phosphorylation and the stabilization of cell surface EGFR. These defects were complemented by wild type Rin1 expression but not by mutant Rin1 lacking a functional Vps9 domain, suggesting that the GEF activity of Rin1 is necessary for proper EGFR signaling in A549 cells. In addition, overexpression of Rin1Δ, dominant negative Rab5, and dynamin resulted in similar defects in cell proliferation and EGFR signaling as Rin1 depletion. These data indicate that proper EGFR internalization and trafficking are critical for robust EGFR-mediated signaling and cell proliferation in A549 cells and offer evidence that Rin1 positively regulates cell proliferation in non-small cell lung adenocarcinoma.     

Thermal biology and establishment potential in temperate climates of the predatory mirid <Emphasis Type="Italic">Nesidiocoris tenuis</Emphasis>

Nesidiocoris tenuis Reuter (Hemiptera: Miridae) is a polyphagous mirid currently used for the control of leafminers, thrips, whitefly and spider mites in Mediterranean regions to which it is indigenous. This study investigates the establishment potential of N. tenuis in cool temperate climates typical of northern Europe through assessment of its thermal biology and low temperature tolerance in laboratory and field experiments. The developmental threshold of N. tenuis was estimated to be 12.9°C with no indication of ability to diapause. Supercooling points of the acclimated and non-acclimated adults and nymphs of the mirid were between −17.6° and −21.5°C and the LTemp₅₀ was around −12°C, indicating a high level of pre-freeze mortality. The LTime₅₀ at 5°C was nine days and 100% mortality occurred after less than four weeks of winter field exposure. Collectively these data suggest that N. tenuis is unlikely to establish in northern Europe and would therefore have little or no non-target effects on native species in such regions.     

Thermal activity thresholds of the predatory mirid Nesidiocoris tenuis: implications for its efficacy as a biological control agent

This study investigates the thermal activity thresholds of the predatory mirid Nesidiocoris tenuis Reuter (Hemiptera: Miridae) and two spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae). Adult N. tenuis lost locomotory function and entered chill coma at significantly lower temperatures (4.0°C and 0.3°C, respectively) than adult T. urticae (7.0°C and 5.7°C, respectively). However, the mirids were more adversely affected by high temperatures, with T. urticae losing the ability to walk and entering heat coma at higher temperatures (47.3°C and 49.7°C, respectively) than N. tenuis (43.5°C and 46.6°C, respectively). Across a range of temperatures (2.5–20°C) adult N. tenuis had faster walking speeds than T. urticae. These data are discussed in relation to the climatic conditions under which N. tenuis would be an effective biocontrol agent.     

Exquisite binding specificity of <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> lectin toward TF-related O-linked mucin-type glycans

Sclerotium rolfsii lectin (SRL), a secretory protein from the soil borne phytopathogenic fungus Sclerotium rolfsii, has shown in our previous studies to bind strongly to the oncofetal Thomson-Friedenreich carbohydrate (Galβ1-3GalNAc-ser/thr, T or TF) antigen. TF antigen is widely expressed in many types of human cancers and the strong binding of SRL toward such a cancer-associated carbohydrate structure led us to characterize the carbohydrate binding specificity of SRL. Glycan array analysis, which included 285 glycans, shows exclusive binding of SRL to the O-linked mucin type but not N-linked glycans and amongst the mucin type O-glycans, lectin recognizes only mucin core 1, core 2 and weakly core 8 but not to other mucin core structures. It binds with high specificity to “α-anomers” but not the “β-anomers” of the TF structure. The axial C4-OH group of GalNAc and C2-OH group of Gal is both essential for SRL interaction with TF disaccharide, and substitution on C3 of galactose by sulfate or sialic acid or N-acetylglucosamine, significantly enhances the avidity of the lectin. SRL differs in its binding to TF structures compared to other known TF-binding lectins such as the Arachis hypogea (peanut) agglutinin, Agaricus bisporus (mushroom) lectin, Jackfruit, Artocarpus integrifolia (jacalin) and Amaranthus caudatus (Amaranthin) lectin. Thus, SRL has unique carbohydrate-binding specificity toward TF-related O-linked carbohydrate structures. Such a binding specificity will make this lectin a very useful tool in future structural as well as functional analysis of the cellular glycans in cancer studies.     

Diversity and distribution of a key sulpholipid biosynthetic gene in marine microbial assemblages

Sulphoquinovosyldiacylglycerols (SQDG) are polar sulphur‐containing membrane lipids, whose presence has been related to a microbial strategy to adapt to phosphate deprivation. In this study, we have targeted the sqdB gene coding the uridine 5′‐diphosphate‐sulphoquinovose (UDP‐SQ) synthase involved in the SQDG biosynthetic pathway to assess potential microbial sources of SQDGs in the marine environment. The phylogeny of the sqdB‐coding protein reveals two distinct clusters: one including green algae, higher plants and cyanobacteria, and another one comprising mainly non‐photosynthetic bacteria, as well as other cyanobacteria and algal groups. Evolutionary analysis suggests that the appearance of UDP‐SQ synthase occurred twice in cyanobacterial evolution, and one of those branches led to the diversification of the protein in members of the phylum Proteobacteria. A search of homologues of sqdB‐proteins in marine metagenomes strongly suggested the presence of heterotrophic bacteria potential SQDG producers. Application of newly developed sqdB gene primers in the marine environment revealed a high diversity of sequences affiliated to cyanobacteria and Proteobacteria in microbial mats, while in North Sea surface water, most of the detected sqdB genes were attributed to the cyanobacterium Synechococcus sp. Lipid analysis revealed that specific SQDGs were characteristic of microbial mat depth, suggesting that SQDG lipids are associated with specific producers.     

Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice

Several microRNAs have been implicated in neurogenesis, neuronal differentiation, neurodevelopment, and memory. Development of miRNA-based therapeutics, however, needs tools for effective miRNA modulation, tissue-specific delivery, and in vivo evidence of functional effects following the knockdown of miRNA. Expression of miR-29a is reduced in patients and animal models of several neurodegenerative disorders, including Alzheimer''s disease, Huntington''s disease, and spinocerebellar ataxias. The temporal expression pattern of miR-29b during development also correlates with its protective role in neuronal survival. Here, we report the cellular and behavioral effect of in vivo, brain-specific knockdown of miR-29. We delivered specific anti-miRNAs to the mouse brain using a neurotropic peptide, thus overcoming the blood-brain-barrier and restricting the effect of knockdown to the neuronal cells. Large regions of the hippocampus and cerebellum showed massive cell death, reiterating the role of miR-29 in neuronal survival. The mice showed characteristic features of ataxia, including reduced step length. However, the apoptotic targets of miR-29, such as Puma, Bim, Bak, or Bace1, failed to show expected levels of up-regulation in mice, following knockdown of miR-29. In contrast, another miR-29 target, voltage-dependent anion channel1 (VDAC1), was found to be induced several fold in the hippocampus, cerebellum, and cortex of mice following miRNA knockdown. Partial restoration of apoptosis was achieved by down-regulation of VDAC1 in miR-29 knockdown cells. Our study suggests that regulation of VDAC1 expression by miR-29 is an important determinant of neuronal cell survival in the brain. Loss of miR-29 results in dysregulation of VDAC1, neuronal cell death, and an ataxic phenotype.     

Difficulties in location and acceptance of phloem sap combined with reduced concentration of phloem amino acids explain lowered performance of the aphid Rhopalosiphum padi on nitrogen deficient barley (Hordeum vulgare) seedlings

Effects of nitrogen deficiency in hydroponically grown barley seedlings (Hordeum vulgare L.) on the development and reproduction of the aphid Rhopalosiphum padi (L.) (Hemiptera: Aphididae) were investigated.Plant growth was significantly reduced in seedlings grown without nitrogen. Aphid intrinsic rate of increase (r _m) was also significantly lower on these plants compared with that on plants grown with 8 mol m^–3 nitrogen. Phloem sap was collected from seedling stems by aphid stylectomy and amino acids quantified by HPLC. There was a significant reduction in the concentration of non-essential amino acids as a group, but not of essential amino acids. Electrical penetration graphs (EPG) indicated that aphids reached the phloem more quickly and fed for longer on plants grown with nitrogen. This is the first reported study in which this combination of techniques has been used to understand the interactions of an aphid and plant under different environmental conditions.     

Incorporation of thrombospondin into fibrin clots

Thrombospondin is a major platelet glycoprotein which is released from platelets during blood coagulation. We examined the interaction of thrombospondin with polymerizing fibrin. Thrombospondin, purified from human platelets and labeled with 125I, became incorporated into clots formed from both plasma and purified fibrinogen. Plasma clots contained somewhat less thrombospondin than clots formed from equivalent concentrations of fibrinogen. In plasma clots and fibrin clots formed in the presence of factor XIII, thrombospondin was cross-linked in the clot; thrombospondin in the supernatant remained largely monomeric. Cross-linking of thrombospondin by factor XIII, however, only slightly increased the amount of thrombospondin which was incorporated into the clot. In contrast, incorporation of 125I-fibronectin into clots was dependent upon cross-linking. Most of the incorporation of 125I-thrombospondin occurred during fibrin polymerization as judged by parallel studies of the incorporation of 125I-fibrinogen. The amount of thrombospondin incorporated into a clot was directly related to thrombospondin concentration and was only weakly dependent on fibrinogen concentration. Incorporation was not saturated at thrombospondin:fibrin (mol/mol) ratios as high as 2/1. Thrombospondin, however, modified the final structure of fibrin clots in a concentration-dependent manner as monitored by opacity. When tryptic digests of 125I-thrombospondin were studied, the 270-kilodalton core became incorporated into fibrin whereas the 30-kilodalton heparin binding fragment was excluded. These results indicate that thrombospondin specifically co-polymerizes with fibrin during blood coagulation and may be an important modulator of clot structure.