N-3 polyunsaturated fatty acids promote astrocyte differentiation and neurotrophin production independent of cAMP in patient-derived neural stem cells.

Evidence from epidemiological and laboratory studies, as well as randomized placebo-controlled trials, suggests supplementation with n-3 polyunsaturated fatty acids (PUFAs) may be efficacious for treatment of major depressive disorder (MDD). The mechanisms underlying n-3 PUFAs potential therapeutic properties remain unknown. There are suggestions in the literature that glial hypofunction is associated with depressive symptoms and that antidepressants may normalize glial function. In this study, induced pluripotent stem cells (iPSC)-derived neuronal stem cell lines were generated from individuals with MDD. Astrocytes differentiated from patient-derived neuronal stem cells (iNSCs) were verified by GFAP. Cells were treated with eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or stearic acid (SA). During astrocyte differentiation, we found that n-3 PUFAs increased GFAP expression and GFAP positive cell formation. BDNF and GDNF production were increased in the astrocytes derived from patients subsequent to n-3 PUFA treatment. Stearic Acid (SA) treatment did not have this effect. CREB activity (phosphorylated CREB) was also increased by DHA and EPA but not by SA. Furthermore, when these astrocytes were treated with n-3 PUFAs, the cAMP antagonist, RP-cAMPs did not block n-3 PUFA CREB activation. However, the CREB specific inhibitor (666-15) diminished BDNF and GDNF production induced by n-3 PUFA, suggesting CREB dependence. Together, these results suggested that n-3 PUFAs facilitate astrocyte differentiation, and may mimic effects of some antidepressants by increasing production of neurotrophic factors. The CREB-dependence and cAMP independence of this process suggests a manner in which n-3 PUFA could augment antidepressant effects. These data also suggest a role for astrocytes in both MDD and antidepressant action.

A comparative analysis of gut microbiota disturbances in the Gottingen minipig and rhesus macaque models of acute radiation syndrome following bioequivalent radiation exposures.

In rodent studies, the gut microbiota has been implicated in facilitating both radioresistance, by protecting the epithelium from apoptotic responses and radiosensitivity, inducing endothelial apoptotic responses. Despite the observation that large animal models, such as the Chinese Rhesus macaque and the Gottingen Minipig, demonstrate similarity to human physiologic responses to radiation, little is known about radiation-induced changes of the gut microbiome in these models. To compare the two models, we used bioequivalent radiation doses which resulted in an LD50 for Gottingen Minipigs and Chinese Rhesus macaques, 1.9 Gy and 6.8 Gy, respectively. Fecal samples taken prior and 3 days post-radiation were used for 16S rRNA gene sequence amplicon high throughput sequencing (Illumina MiSeq). Baseline gut microbiota profiles were dissimilar between minipigs and rhesus macaques. Irradiation profoundly impacted gut microbiota profiles in both animals. Significant increases of intracellular symbionts were common to both models and to reported changes in rodents suggesting universality of these findings post-radiation. Remarkably, opposite dynamics were observed for the main phyla, with increase of Firmicutes and decrease of Bacteroidetes and Proteobacteria in minipigs but with enrichment of Bacteroidetes in rhesus macaques. Minipig changes in magnitude and in variety of species affected were more extensive than those observed in rhesus macaques. This pilot study provides an important first step in comparing the radiosensitive pig model to the comparatively more radioresistant macaque model, for the identification of microbial elements which may influence radiosensitivity.

Genome-Wide Association Analysis Identifies Important Haplotypes and Candidate Gene XKR4 for Body Size Traits in Pekin Ducks.

Body size is an important growth indicator in ducks and is a primary selection criterion for physical improvement. An excessively rapid growth rate in meat ducks can result in excessive body size, which may hinder subsequent processing and slaughter operations. However, only a few molecular markers related to body size have been studied in meat ducks. In this study, we performed a genome-wide association study (GWAS) to identify candidate genes and QTLs affecting body length (BL), keel bone length (KBL), neck length (NL), and breast width (BrW) in Pekin ducks (Anas platyrhynchos domestica). Our results indicate the significant SNP for NL is located within a pseudogene, whereas the significant SNP for BrW is located in an intergenic region. More importantly, our analysis identified a haplotype that was significantly associated with both BL and KBL. This haplotype, containing 48 single-nucleotide polymorphisms (SNPs), is localized within the XKR4 gene. The identification of this haplotype suggests that XKR4 may be a key candidate gene influencing BL and KBL in Pekin ducks. These findings have important implications for the breeding and genetic improvement of Pekin ducks, and provide valuable insights into the genetic architecture of body size traits in this species.

Genome-Wide Association Analysis and Genetic Parameters for Egg Production Traits in Peking Ducks.

Egg production traits are crucial in the poultry industry, including age at first egg (AFE), egg number (EN) at different stages, and laying rate (LR). Ducks exhibit higher egg production capacity than other poultry species, but the genetic mechanisms are still poorly understood. In this study, we collected egg-laying data of 618 Peking ducks from 22 to 66 weeks of age and genotyped them by whole-genome resequencing. Genetic parameters were calculated based on SNPs, and a genome-wide association study (GWAS) was performed for these traits. The SNP-based heritability of egg production traits ranged from 0.09 to 0.54. The GWAS identified nine significant SNP loci associated with AFE and egg number from 22 to 66 weeks. These loci showed that the corresponding alleles were positively correlated with a decrease in the traits. Moreover, three potential candidate genes (ENSAPLG00020011445, ENSAPLG00020012564, TMEM260) were identified. Functional enrichment analyses suggest that specific immune responses may have a critical impact on egg production capacity by influencing ovarian function and oocyte maturation processes. In conclusion, this study deepens the understanding of egg-laying genetics in Peking duck and provides a sound theoretical basis for future genetic improvement and genomic selection strategies in poultry.

Heterotrimeric G-proteins interact directly with cytoskeletal components to modify microtubule-dependent cellular processes.

A large percentage of current drugs target G-protein-coupled receptors, which couple to well-known signaling pathways involving cAMP or calcium. G-proteins themselves may subserve a second messenger function. Here, we review the role of tubulin and microtubules in directly mediating effects of heterotrimeric G-proteins on neuronal outgrowth, shape and differentiation. G-protein-tubulin interactions appear to be regulated by neurotransmitter activity, and, in turn, regulate the location of Galpha in membrane microdomains (such as lipid rafts) or the cytosol. Tubulin binds with nanomolar affinity to Gsalpha, Gialpha1 and Gqalpha (but not other Galpha subunits) as well as Gbeta(1)gamma(2) subunits. Galpha subunits destabilize microtubules by stimulating tubulin's GTPase, while Gbetagamma subunits promote microtubule stability. The same region on Gsalpha that binds adenylyl cyclase and Gbetagamma also interacts with tubulin, suggesting that cytoskeletal proteins are novel Galpha effectors. Additionally, intracellular Gialpha-GDP, in concert with other GTPase proteins and Gbetagamma, regulates the position of the mitotic spindle in mitosis. Thus, G-protein activation modulates cell growth and differentiation by directly altering microtubule stability. Further studies are needed to fully establish a structural mechanism of this interaction and its role in synaptic plasticity.

Specific Members of the Gut Microbiota are Reliable Biomarkers of Irradiation Intensity and Lethality in Large Animal Models of Human Health.

The development of effective biomarkers for detecting the magnitude of radiation exposure and resiliency of host response is crucial to identifying appropriate treatment strategies after radiation exposure. We hypothesized that the gastrointestinal resident bacteria would demonstrate predictable, dose-dependent changes after radiation exposure across two large animal models of acute radiation syndrome. Here, Göttingen minipigs (GMP) (n = 50) and rhesus macaques (n = 48) were exposed to five dose levels (resulting in mortality rates of 33-100% and 25-68.7%, respectively). Fecal samples taken prior to and after irradiation (day 0 for GMP; day 0, 3 and 14 for macaques) were used for 16S rRNA gene sequence amplicon high-throughput sequencing. Baseline gut microbiota profiles were dissimilar between GMP and macaques, however, radiation appeared to have similar effect at the phylum level, resulting in Bacteroidetes decrease and Firmicutes increase in both models. The abundance of the main Bacteroidetes genus ( Bacteroides for GMP, Prevotella for macaques) was profoundly decreased by irradiation. Intracellular symbionts [Elusimicrobia in GMP, Treponema (Spirochaetes) in macaques] consistently increased after irradiation, suggesting their use as potential biomarkers of intestinal injury, and potential negative effect on health. Prevotella, Lactobacillus, Clostridium XIVa, Oscillibacter and Elusimicrobium/ Treponema abundances were found to be very significantly correlated with radiation intensity. Furthermore, Prevotella, Enterorhabdus and Ruminococcus and Enterorhabdus maintenance was strongly associated with survival in GMP, while Prevotella, Oscillibacter and Treponema were strongly associated with survival and Streptococcus with death in macaques. Overall, we found that a wide range of gut bacterial genera known to be abundant in the human gut microbiota are excellent biomarkers of radiation intensity and resilience in animal models, and that detrimental effects can be monitored, and potentially prevented, by targeting selected genera.

Mastoparan inhibits beta-adrenoceptor-G(s) signaling by changing the localization of Galpha(s) in lipid rafts.

Mastoparan, a wasp venom toxin, has various pharmacological activities, the mechanisms of which are still unknown. To clarify the action of mastoparan on G protein-coupled receptor-mediated signaling, we previously examined the effect of mastoparan on G(q)-mediated signaling and demonstrated that mastoparan binds to gangliosides causing a decrease in Galpha(q/11) content in lipid rafts, and resulting in the inhibition of G(q)-mediated phosphoinositide hydrolysis (Sugama et al., Mol. Pharmacol., 68, 1466, 2005). In the present study, we examined the effect of mastoparan on beta-adrenoceptor-G(s) signaling in 1321N1 human astrocytoma cells. Mastoparan inhibited isoproterenol-induced elevation of cyclic AMP in a concentration-dependent manner. Although mastoparan is known to be an activator of G(i), pertussis toxin only slightly attenuated mastoparan-induced inhibition of cyclic AMP elevation, suggesting that a major part of the inhibition of cyclic AMP elevation induced by mastoparan is not mediated by Galpha(i). By contrast, mastoparan-induced inhibition of cyclic AMP elevation was clearly attenuated by preincubation of the cells with ganglioside mixtures. Moreover, mastoparan changed the localization of Galpha(s) in lipid rafts without disrupting the structure of lipid rafts. Fluorescent staining analysis showed that mastoparan released GFP-Galpha(s) from plasma membranes into the cytosol. These results suggest that the mastoparan-induced suppression of cyclic AMP elevation is mainly caused by changing the localization of Galpha(s) in lipid rafts into a compartment in the cellular interior where it is not available to activate adenylyl cyclase.

Tau associates with actin in differentiating PC12 cells.

The microtubule-associated protein tau may be involved in cell morphogenesis and axonal maintenance. In addition to microtubules, tau has been shown to interact with actin in vitro. In the present study interaction of tau and actin was investigated in PC12 cells. No interaction between tau and actin was observed without NGF treatment. Under NGF stimulation, tau distributed at ends of cellular extensions, where it associated with actin in a microtubule-independent manner. F-actin disruption revealed that relocalization and assembly of F-actin at the ends of cellular extensions were necessary for NGF-induced tau reorganization and association with actin. A truncated tau-GFP (tau(1-186)-GFP, N-terminal of tau) did not associate with actin. However, tau23(174-352)-GFP (carboxyl-terminal of Tau23) did associate with actin and the requirement for NGF was lost. Nevertheless, NGF boosted tau23(174-352)-GFP interaction with actin and promoted colocalization at the ends of cellular extensions. This suggests that the C-terminal of tau is required for associating with actin and the tau N-terminal may play a regulatory role in this process. A possible role for tau-actin interaction in neurite outgrowth is postulated.

Subject-Based versus Population-Based Care after Radiation Exposure.

In a mass casualty radiation event situation, individualized therapy may overwhelm available resources and feasibility issues suggest a need for the development of population-based strategies. To investigate the efficacy of a population-based strategy, Chinese macaques (n = 46) underwent total-body irradiation and received preemptive antibiotics, IV hydration on predetermined postirradiation days and were then compared to macaques (n = 48) that received subject-based care in which blood transfusions, IV hydration, nutritional supplementation and antibiotic supportive measures were provided. Estimated radiation doses for LD30/60, LD50/60 and LD70/60 of animals with subject-based care: 6.83 Gy (6.21, 7.59), 7.44 Gy (6.99, 7.88) and 8.05 Gy (7.46, 8.64), respectively, and for population-based care: 5.61 Gy (5.28, 6.17), 6.62 Gy (6.13, 7.18) and 7.63 Gy (7.21, 8.20), respectively. Analysis of four time periods, 0-9, 10-15, 16-25 and 26-60 days postirradiation, identified significant mortality differences during the period of 10-15 days. A subset analysis of higher radiation doses (6.75-7.20 Gy, n = 32) indicated hydration, nutrition and septic status were not significantly different between treatments. Whole blood transfusion treatment, administered only in subject-supportive care, was associated with significantly higher platelet and absolute neutrophil counts. Median platelet counts greater than 5,670 cells/µl and absolute neutrophil counts greater than 26 cells/µl during this period correlated with survival. We observed that the population-based treatment increased the LD50/60 compared to nontreatment (6.62 Gy vs. 4.92 Gy) and may be further optimized during days 10-15, where strategic blood transfusions or other strategies to achieve increases in neutrophil and platelet counts may further increase survival rates in subjects exposed to high doses of radiation.

Tubulin as a regulator of G-protein signaling.

Tubulin is known to form high-affinity complexes with certain G proteins. The formation of such complexes allows tubulin to activate Galpha and fosters a system whereby elements of the cytoskeleton can influence G-protein signaling. This article describes the interaction between tubulin and G proteins and discusses methods for examining this interaction.

Receptor signaling and the cell biology of synaptic transmission.

This volume describes a series of psychiatric and neuropsychiatric disorders, connects some aspects of somatic and psychiatric medicine, and describes various current and emerging therapies. The purpose of this chapter is to set the stage for the volume by developing the theoretical basis of synaptic transmission and introducing the various neurotransmitters and their receptors involved in the process. The intent is to provide not only a historical context through which to understand neurotransmitters, but a current contextual basis for understanding neuronal signal transduction and applying this knowledge to facilitate treatment of maladies of the brain and mind.

Cytosolic G{alpha}s acts as an intracellular messenger to increase microtubule dynamics and promote neurite outgrowth.

It is now evident that Galpha(s) traffics into cytosol following G protein-coupled receptor activation, and alpha subunits of some heterotrimeric G-proteins, including Galpha(s) bind to tubulin in vitro. Nevertheless, many features of G-protein-microtubule interaction and possible intracellular effects of G protein alpha subunits remain unclear. In this study, several biochemical approaches demonstrated that activated Galpha(s) directly bound to tubulin and cellular microtubules, and fluorescence microscopy showed that cholera toxin-activated Galpha(s) colocalized with microtubules. The activated, GTP-bound, Galpha(s) mimicked tubulin in serving as a GTPase activator for beta-tubulin. As a result, activated Galpha(s) made microtubules more dynamic, both in vitro and in cells, decreasing the pool of insoluble microtubules without changing total cellular tubulin content. The amount of acetylated tubulin (an indicator of microtubule stability) was reduced in the presence of Galpha(s) activated by mutation. Previous studies showed that cholera toxin and cAMP analogs may stimulate neurite outgrowth in PC12 cells. However, in this study, overexpression of a constitutively activated Galpha(s) or activation of Galpha(s) with cholera toxin in protein kinase A-deficient PC12 cells promoted neurite outgrowth in a cAMP-independent manner. Thus, it is suggested that activated Galpha(s) acts as an intracellular messenger to regulate directly microtubule dynamics and promote neurite outgrowth. These data serve to link G-protein signaling with modulation of the cytoskeleton and cell morphology.

Real-time visualization of a fluorescent G(alpha)(s): dissociation of the activated G protein from plasma membrane.

To study behavior of activated G(alpha)(s) in living cells, green fluorescent protein (GFP) was inserted within the internal amino acid sequence of G(alpha)(s) to generate a G(alpha)(s)-GFP fusion protein. The fusion protein maintained a bright green fluorescence and was identified by immunoblotting with antibodies against G(alpha)(s) or GFP. The cellular distribution of G(alpha)(s)-GFP was similar to that of endogenous G(alpha)(s). G(alpha)(s)-GFP was tightly coupled to the beta adrenergic receptor to activate the G(alpha)(s) effector, adenylyl cyclase. Activation of G(alpha)(s)-GFP by cholera toxin caused a gradual displacement of the fusion protein from the plasma membrane throughout the cytoplasm in living cells. Unlike the slow release of G(alpha)(s)-GFP from the membrane induced by cholera toxin, the beta-adrenergic agonist isoproterenol caused a rapid partial release of the fusion protein into the cytoplasm. At 1 min after treatment with isoproterenol, the extent of G(alpha)(s)-GFP release from plasma membrane sites was maximal; however, insertion of G(alpha)(s)-GFP at other membrane sites occurred during the same time period. Translocation of G(alpha)(s)-GFP fusion protein induced by isoproterenol suggested that the internalization of G(alpha)(s) might play a role in signal transduction by interacting with effector molecules and cytoskeletal elements at multiple cellular sites.

Transient expression of fluorescent tau proteins promotes process formation in PC12 cells: contributions of the tau C-terminus to this process.

The neuronal microtubule-associated protein tau promotes microtubule assembly and has been implicated in the development of axonal morphology. In this study, PC12 cells were transiently transfected with constructs coding fusion proteins of human tau with green fluorescent protein (GFP). Expression of tau constructs actively stabilized microtubules. Expression of the C-terminus of tau can mimic this effect in living cells, though to a lesser extent because of the absence of the tau N-terminus. However, tau colocalization with microtubules did not require the presence of the tau N-terminus. Transient expression of tau (including tau24, a four-repeat human tau isoform encoded in 383 residues, and tau23, human fetal tau isoform encoded in 352 residues) stimulated process formation in PC12 cells, and this occurred faster with tau24 than with tau23. The residues (residues 154-172 in tau23) that confer microtubule nucleation activity of tau in vitro are not required for tau-directed process formation. However, when tau induces the formation of cellular processes in response to cortical breakdown by cytochalasin B, residues 154-172 must be present. Thus, it appears that tau may serve to promote cellular process outgrowth in cultured neuronal cells and that C-terminus of tau is essential to this process.

Tau interaction with microtubules in vivo.

Tau is a major microtubule-associated protein which induces bundling and stabilization of axonal microtubules (MTs). To investigate the interaction of tau with MTs in living cells, we expressed GFP-tau fusion protein in cultured Xenopus embryo neurons and performed time-lapse imaging of tau-labeled MTs. Tau uniformly labeled individual MTs regardless of their assembly/disassembly status and location along the axon. Photobleaching experiments indicated that interaction of tau with MTs is very dynamic, with a half-time of fluorescence recovery of the order of 3 seconds. Treatment of cells with taxol, a drug that suppresses MT dynamics, rapidly induced detachment of tau from MTs. Although binding of tau to straight MTs was uniform, there was a heightened concentration of tau at the sites of high MT curvature. Our results suggest that dynamic interaction of tau with MTs may modify local mechanical properties of individual MTs and play a crucial role in the remodeling of the MT cytoskeleton during neuronal plasticity.

A specific domain of Gialpha required for the transactivation of Gialpha by tubulin is implicated in the organization of cellular microtubules.

G(s)alpha, G(i)alpha(1), and G(q)alpha subunits bind tubulin with high affinity, whereas transducin (G(t)alpha) does not. The interaction between tubulin and Galpha, which also involves the direct transfer of GTP from tubulin to Galpha (transactivation), is not yet fully understood. This study, using chimeras of G(i)alpha and G(t)alpha, showed that the G(i)alpha (215-295) segment converted G(t)alpha to bind to tubulin and this chimera (chimera 1) could be transactivated by tubulin. Insertion of G(t)alpha (237-270) into chimera 1 to form chimera 2 resulted in a protein that, like G(t)alpha, did not bind tubulin. Thus, it was thought that the G(i)alpha (237-270) domain was essential to modulate the binding of G(i)alpha(1) to tubulin. Surprisingly, when domain (237-270) of G(i)alpha was replaced by G(t)alpha (237-270) to form chimera 3, the chimera bound to tubulin with a similar affinity (K(D) congruent with 120 nm) as wild-type G(i)alpha(1). However, even though chimera 3 displayed normal GTP binding, it was not transactivated by GTP-tubulin. Furthermore, when these chimeras were expressed in COS-1 cells, cellular processes in cells overexpressing G(i)alpha(1) or chimera 1 were more abundant and longer than those in native cells. Galpha was seen throughout the length of the process. Morphology of cells expressing chimera 2 was identical to controls. Consistent with the role of Chimera 3 as a "dominant negative" Galpha, cells transfected with chimera 3 had only few truncated processes. This study demonstrates that although G(i)alpha (237-270) is not obligatory for the binding of G(i)alpha to tubulin, it is crucial for the transactivation of Galpha by tubulin. These results also suggest that the transactivation of Galpha by tubulin may play an important role in modulating microtubule organization and cell morphology.