Reproduction Immunity Trade-Off in a Mollusk: Hemocyte Energy Metabolism Underlies Cellular and Molecular Immune Responses

Immune responses, as well as reproduction, are energy-hungry processes, particularly in broadcast spawners such as scallops. Thus, we aimed to explore the potential reproduction-immunity trade-off in Argopecten purpuratus, a species with great economic importance for Chile and Peru. Hemocytes, key immunological cells in mollusks, were the center of this study, where we addressed for the first time the relation between reproductive stage, hemocyte metabolic energetics and their capacity to support immune responses at cellular and molecular levels. Hemocyte metabolic capacity was assessed by their respiration rates, mitochondrial membrane potential and citrate synthase (CS) activity. Cellular immune parameters such as the number of circulating and tissue-infiltrating hemocytes and their reactive oxygen species (ROS) production capacity were considered. Molecular immune responses were examined through the transcriptional levels of two pattern recognition receptors (ApCLec and ApTLR) and two anti-microbial effectors (ferritin and big defensin). Their expressions were measured in hemocytes from immature, matured and spawned scallops under basal, and one of the following challenges: (i) in vitro, where hemocytes were challenged with the β glucan zymosan, to determine the immune potentiality under standardized conditions; or (ii) in vivo challenge, using hemocytes from scallops injected with the pathogenic bacteria Vibrio splendidus. Results indicate a post-spawning decrease in the structural components of the immune system (hemocyte number/quality) and their potential capacity of performing immune functions (with reduced ATP-producing machinery and exhaustion of energy reserves). Both in vitro and in vivo challenges demonstrate that hemocytes from immature scallops have, in most cases, the best metabolic potential (increased CS activity) and immune performances, with for example, over threefold higher ROS production and tissue-infiltration capacity than those from mature and spawned scallops after the bacterial challenge. Agreeing with cellular responses, hemocytes from immature individuals induced the highest levels of immune receptors and antimicrobial effectors after the bacterial challenge, while spawned scallops presented the lowest values. Overall, results suggest a trade-off between resource allocation in reproduction and the immune responses in A. purpuratus, with hemocyte energy metabolic capacity potentially underlying cellular and molecular immune responses. Further research would be necessary to explore regulatory mechanisms such as signaling pleiotropy which may potentially be underlying this trade-off

Differential Distribution and Activity Profile of Acylpeptide Hydrolase in the Rat Seminiferous Epithelium

Acylpeptide hydrolase (APEH) is a serine protease involved in amino acid recycling from acylated peptides (exopeptidase activity) and degradation of oxidized proteins (endoproteinase activity). This enzyme is inhibited by dichlorvos (DDVP), an organophosphate compound used as an insecticide. The role of APEH in spermatogenesis has not been established; therefore, the aim of this study was to characterize the distribution and activity profile of APEH during this process. For this purpose, cryosections of male reproductive tissues (testis and epididymis) and isolated cells (Sertoli cells, germ cells, and spermatozoa) were obtained from adult rats in order to analyze the intracellular localization of APEH by indirect immunofluorescence. In addition, the catalytic activity profiles of APEH in the different male reproductive tissues and isolated cells were quantified. Our results show that APEH is homogeneously distributed in Sertoli cells and early germ cells (spermatocytes and round spermatids), but this pattern changes during spermiogenesis. Specifically, in elongated spermatids and spermatozoa, APEH was localized in the acrosome and the principal piece. The exopeptidase activity was higher in the germ cell pool, compared to sperm and Sertoli cells, while the endoproteinase activity in epididymal homogenates was higher compared to testis homogenates at 24 h of incubation. In isolated cells, this activity was increased in Sertoli and germ cell pools, compared to spermatozoa. Taken together, these results indicate that APEH is differentially distributed in the testicular epithelium and undergoes re-localization during spermiogenesis. A possible role of APEH as a component of a protection system against oxidative stress and during sperm capacitation is discussed

NADPH Oxidase Isoform 2 (NOX2) Is Involved in Drug Addiction Vulnerability in Progeny Developmentally Exposed to Ethanol

Ethanol exposure increases oxidative stress in developing organs, including the brain. Antioxidant treatment during maternal ethanol ingestion improves behavioral deficits in rodent models of fetal alcohol spectrum disorder (FASD). However, the impact of general antioxidant treatment in their adult offspring and the Specific Reactive Species (ROS)-dependent mechanism, are not fully understood. We hypothesized that pre and early postnatal ethanol exposure (PEE) modifies redox homeostasis, in particular NOX2 function during reward signaling in the mesocorticolimbic pathway, which reinforces the effects of alcohol. We developed a FASD rat model which was evaluated during adolescence (P21) and adulthood (P70). We first studied whether redox homeostasis is affected in PEE animals, by analyzing mRNA expression of SOD1, CAT, and Gpx1. We found that PEE reduced the mRNA levels of these three anti-oxidant enzymes in PFC and HIPP at P21 and in the VTA at P70. We also analyzed basal mRNA and protein expression of NOX2 subunits such as gp91phox, p22 phox, and p47 phox, in mesocorticolimbic brain areas of PEE rat brains. At P21, gp91 phox, and p47 phox levels in the VTA were decreased. At P70, gp91 phox mRNA levels was decreased in HIPP and both mRNA and protein levels were decreased in PFC. Since NOX2 is regulated by the N-methyl-D-aspartate Receptor (NMDAR), we analyzed NMDAR mRNA expression and found differential expression of NMDAR subunits (NR1 and NR2B) in the PFC that was age dependent, with levels decreased at P21 and increased at P70. The analysis also revealed decreased NR2B mRNA expression in HIPP and VTA at P70. Offspring from maternal ethanol users consumed 25% more ethanol in a free choice alcohol consumption test than control rats, and showed place preference for an alcohol-paired compartment. In vivo inhibition of NOX2 using apocynin in drinking water, or infusion of blocked peptide gp91 phox ds in the VTA normalized alcohol place preference, suggesting that NOX2 plays an important role in addictive like behavior. Taken together, PEE significantly affects the expression of antioxidant enzymes, NOX2, NMDAR in an age, and brain region dependent manner. Moreover, we demonstrate that NOX2 regulates alcohol seeking behavior

Purinergic P2Y2 and P2X4 Receptors Are Involved in the Epithelial-Mesenchymal Transition and Metastatic Potential of Gastric Cancer Derived Cell Lines

Gastric cancer (GC) is a major health concern worldwide, presenting a complex pathophysiology that has hindered many therapeutic efforts so far. In this context, purinergic signaling emerges as a promising pathway for intervention due to its known role in cancer cell proliferation and migration. In this work, we explored in more detail the role of purinergic signaling in GC with several experimental approaches. First, we measured extracellular ATP concentrations on GC-derived cell lines (AGS, MKN-45, and MKN-74), finding higher levels of extracellular ATP than those obtained for the non-tumoral gastric cell line GES-1. Next, we established the P2Y2 and P2X4 receptors (P2Y2R and P2X4R) expression profile on these cells and evaluated their role on cell proliferation and migration after applying overexpression and knockdown strategies. In general, a P2Y2R overexpression and P2X4R downregulation pattern were observed on GC cell lines, and when these patterns were modified, concomitant changes in cell viability were observed. These modifications on gene expression also modified transepithelial electrical resistance (TEER), showing that higher P2Y2R levels decreased TEER, and high P2X4R expression had the opposite effect, suggesting that P2Y2R and P2X4R activation could promote and suppress epithelial-mesenchymal transition (EMT), respectively. These effects were confirmed after treating AGS cells with UTP, a P2Y2R-agonist that modified the expression patterns towards mesenchymal markers. To further characterize the effects of P2Y2R activation on EMT, we used cDNA microarrays and observed that UTP induced important transcriptional changes on several cell processes like cell proliferation induction, apoptosis inhibition, cell differentiation induction, and cell adhesion reduction. These results suggest that purinergic signaling plays a complex role in GC pathophysiology, and changes in purinergic balance can trigger tumorigenesis in non-tumoral gastric cells

N-Methyl-d-Aspartate receptor modulation by Nicotinamide Adenine Dinucleotide Phosphate Oxidase type 2 drives synaptic plasticity and spatial memory impairments in rats exposed Pre- and postnatally to Ethanol

Aims: Pre- and/or early postnatal ethanol exposure (prenatal alcohol exposure [PAE]) impairs synaptic plasticity as well as memory formation, but the mechanisms underlying these effects remain unclear. Both long-term potentiation (LTP) and spatial memory formation in the hippocampus involve the nicotinamide adenine dinucleotide phosphate oxidase type 2 (NOX2) enzyme. Previous studies have reported that N-methyl-d-aspartate receptor (NMDAR) activation increases NOX2-mediated superoxide generation, resulting in inhibition of NMDAR function, but whether NOX2 impacts NMDAR function in PAE animals leading to impaired LTP and memory formation remains unknown. We aim to evaluate whether the NOX2-NMDAR complex is involved in the long-lasting deleterious effects of PAE on hippocampal LTP and memory formation. Results: Here we provide novel evidence that PAE animals display impaired NMDAR-dependent LTP in the cornus ammonis field 1 (CA1) and NMDAR-mediated LTP in the dentate gyrus (DG). Moreover, PAE rats displayed increased NMDAR-mediated transmission in both hippocampal areas. Interestingly, NOX2 pharmacological inhibition restored NMDAR-mediated transmission and LTP in the CA1, but not in the DG. PAE also induced overexpression of NOX2 and CaMKII isoforms, but did not modify the content or the redox state of the N-methyl-d-aspartate receptor subunit-1 (NR1) subunit of NMDAR in both areas of the hippocampus. In addition, adolescent PAE rats orally fed the antioxidant and free radical scavenger apocynin exhibited significantly improved spatial memory acquisition. Innovation and Conclusion: By showing in PAE animals NOX2 overexpression and increased NMDAR-mediated transmission, which might lead to impaired synaptic plasticity and memory formation in a region-specific manner, we provide an important advance to our current understanding of the cellular mechanisms underlying PAE-dependent defective hippocampal function.Chilean government through FONDECYT Regular Grant 1140855 1170053 1130407 1151091 Millennium Institute Centro Interdisciplinario de Neurociencia de Valparaiso (CINV) P09-022F Nucleus Biology of Neuropsychiatric Diseases (NuMIND) NC 130011 Millennium Scientific Initiative of the Ministry of Economy, Development and Tourism, Chile Universidad de Valparais

Pre- and postnatal alcohol exposure delays, in female but not in male rats, the extinction of an auditory fear conditioned memory and increases alcohol consumption

Repeated exposure to alcohol increases retrieval of fear-conditioned memories, which facilitates, among other factors, the emergence of post-traumatic stress disorder (PTSD). Individuals with PTSD are more likely to develop alcohol and substance abuse related disorders. We assessed if prenatal and early postnatal alcohol exposure (PAE) increased the susceptibility to retain aversive memories and if this was associated with subsequent heightened alcohol consumption. Pregnant Sprague-Dawley rats were exposed for 22 hr/day, throughout pregnancy and until postnatal Day 7 to a single bottle of sucralose - sweetened 10% alcohol solution (PAE Group), or to a single bottle of tap water and sucralose (Control Group). Auditory fear conditioning (AFC) was performed in the adolescent offspring at postnatal Day 40. Freezing was measured during acquisition, retention and extinction phases, followed by 3 weeks of free choice alcohol intake. Female, but not male, PAE rats exhibited impaired extinction of the aversive memory, a finding associated with higher levels of 3-4 Dihidroxyphenylacetic acid (DOPAC) in the nucleus accumbens and heightened alcohol intake, respect to controls. These findings suggest that PAE makes females more vulnerable to long-term retention of aversive memories, which coexist with heightened alcohol intake. These findings are reminiscent of those of PTSD.Fil: Plaza, Wladimir. Universidad Católica del Norte; ChileFil: Gaschino, Felice. Universidad Católica del Norte; ChileFil: Gutierrez, Camilo. Universidad de Valparaíso; ChileFil: Santibañez, Nicolás. Universidad de Valparaíso; ChileFil: Estay-Olmos, Camila. Universidad de Valparaíso; ChileFil: Sotomayor-Zárate, Ramón. Universidad de Valparaíso; ChileFil: De la Fuente-Ortega, Erwin. Universidad Católica del Norte; ChileFil: Pautassi, Ricardo Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Haeger, Paola A.. Universidad Católica del Norte; Chil

Differential effects of purinergic signaling in gastric cancer-derived cells through P2Y and P2X receptors

Gastric cancer (GC) is the one of the most prevalent cancers and one of the leading causes of cancer-induced deaths. Previously, we found that the expression of purinergic P2Y2\ua0receptor (P2Y2R) is increased in GC samples as compared to adjacent healthy mucosa taken from GC-diagnosed patients. In this work, we studied in detail purinergic signaling in the gastric adenocarcinoma-derived cell lines: AGS, MKN-45, and MKN-74, and compared them to a nontumoral epithelial cell line: GES-1. In GC-derived cells, we detected the expression of several purinergic receptors, and found important differences as compared to GES-1 cells. Functional studies revealed a strong contribution of P2Y2Rs in intracellular calcium increases, elicited by adenosine-triphosphate (ATP), uridine-triphosphate (UTP), and the P2Y2R agonist MRS2768. Responses were preserved in the absence of extracellular calcium and inhibited by P2Y2R antagonists. In GES-1 cells, ATP and UTP induced similar responses and the combination of P2X and P2Y receptor antagonists was able to block them. Proliferation studies showed that ATP regulates AGS and MKN-74 cells in a biphasic manner, increasing cell proliferation at 10–100 μM, but inhibiting at 300 μM ATP. On the other hand, 1–300 μM UTP, a P2Y2R agonist, increased concentration-dependent cell proliferation. The effects of UTP and ATP were prevented by both wide-range and specific purinergic antagonists. In contrast, in GES-1 cells ATP only decreased cell proliferation in a concentration-dependent manner, and UTP had no effect. Notably, the isolated application of purinergic antagonists was sufficient to change the basal proliferation of AGS cells, indicating that nucleotides released by the cells can act as paracrine/autocrine signals. Finally, in tumor-derived biopsies, we found an increase of P2Y2R and a decrease in P2X4R expression; however, we found high variability between seven different biopsies and their respective adjacent healthy gastric mucosa. Even so, we found a correlation between the expression levels of P2Y2R and P2X4R and survival rates of GC patients. Taken together, these results demonstrate the involvement of different purinergic receptors and signaling in GC, and the pattern of expression changes in tumoral cells, and this change likely directs ATP and nucleotide signaling from antiproliferative effects in healthy tissues to proliferative effects in cancer

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present