Perfluoroalkyl acids potentiate glutamate excitotoxicity in rat cerebellar granule neurons.

Perfluoroalkyl acids (PFAAs) are persistent man-made chemicals, ubiquitous in nature and present in human samples. Although restrictions are being introduced, they are still used in industrial processes as well as in consumer products. PFAAs cross the blood-brain-barrier and have been observed to induce adverse neurobehavioural effects in humans and animals as well as adverse effects in neuronal in vitro studies. The sulfonated PFAA perfluorooctane sulfonic acid (PFOS), has been shown to induce excitotoxicity via the N-methyl-D-aspartate receptor (NMDA-R) in cultures of rat cerebellar granule neurons (CGNs). In the present study the aim was to further characterise PFOS-induced toxicity (1-60 µM) in rat CGNs, by examining interactions between PFOS and elements of glutamatergic signalling and excitotoxicity. Effects of the carboxylated PFAA, perfluorooctanoic acid (PFOA, 300-500 µM) on the same endpoints were also examined. During experiments in immature cultures at days in vitro (DIV) 8, PFOS increased both the potency and efficacy of glutamate, whereas in mature cultures at DIV 14 only increased potency was observed. PFOA also increased potency at DIV 14. PFOS-enhanced glutamate toxicity was further antagonised by the competitive NMDA-R antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) at DIV 8. At DIV 8, PFOS also induced glutamate release (9-13 fold increase vs DMSO control) after 1-3 and 24 h exposure, whereas for PFOA a large (80 fold) increase was observed, but only after 24 h. PFOS and PFOA both also increased alanine and decreased serine levels after 24 h exposure. In conclusion, our results indicate that PFOS at concentrations relevant in an occupational setting, may be inducing excitotoxicity, and potentiation of glutamate signalling, via an allosteric action on the NMDA-R or by actions on other elements regulating glutamate release or NMDA-R function. Our results further support our previous findings that PFOS and PFOA at equipotent concentrations induce toxicity via different mechanisms of action.

Novel GTF2I-PDGFRB and IKZF1-TYW1 fusions in pediatric leukemia with normal karyotype.

BACKGROUND: Many cases of acute lymphoblastic leukemia (ALL) carry visible acquired chromosomal changes of pathogenetic, diagnostic, and prognostic importance. Nevertheless, from one-fourth to half of newly diagnosed ALL patients have no visible chromosomal changes detectable by G-banding analysis at diagnosis. The introduction of powerful molecular methodologies has shown that many karyotypically normal ALLs carry clinically important submicroscopic aberrations. CASE PRESENTATION: We used fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH), RNA sequencing, reverse transcription (RT) and genomic polymerase chain reaction (PCR), as well as Sanger sequencing to investigate a case of pediatric ALL with a normal karyotype. FISH with a commercial PDGFRB breakapart probe showed loss of the distal part of the probe suggesting a breakpoint within the PDGFRB locus. aCGH revealed submicroscopic deletions in chromosome bands 5q32q35.3 (about 30 Mb long, starting within PDGFRB and finishing in the CANX locus), 7q34 (within TCRB), 9p13 (PAX5), 10q26.13 (DMBT1), 14q11.2 (TRAC), and 14q32.33 (within the IGH locus). RNA sequencing detected an in-frame GTF2I-PDGFRB and an out-of-frame IKZF1-TYW1 fusion transcript. Both fusion transcripts were verified by RT-PCR together with Sanger sequencing and interphase FISH. The GTF2I-PDGFRB fusion was also verified by genomic PCR and FISH. The corresponding GTF2I-PDGFRB fusion protein would consist of almost the entire GTF2I and that part of PDGFRB which harbors the catalytic domain of the tyrosine kinase. It would therefore seem to lead to abnormal tyrosine kinase activity in a manner similar to what has been seen for other PDGFRB fusion proteins. CONCLUSIONS: The examined pediatric leukemia is a Ph-like ALL which carries novel GTF2I-PDGFRB and IKZF1-TYW1 fusion genes together with additional submicroscopic deletions. Because hematologic neoplasms with PDGFRB-fusion genes can be treated with tyrosine kinase inhibitors, the detection of such novel fusions may be clinically important. Since the GTF2I-PDGFRB could be detected only after molecular studies of the leukemic cells, further investigations of ALL-cases, perhaps especially but not exclusively with a normal karyotype, are needed in order to determine the frequency of GTF2I-PDGFRB in leukemia, and also to find out which clinical impact the fusion may have.

Primary mediastinal choriocarcinoma in a female patient: Case report and review of the literature.

•Primary mediastinal choriocarcinoma is rare, especially in female patients.•Genomic losses predominated our case, which has not been previously reported.•This tumor lacked human chorionic gonadotropin and required histologic diagnosis.

PFOS-induced excitotoxicity is dependent on Ca2+ influx via NMDA receptors in rat cerebellar granule neurons.

Perfluoroalkyl acids (PFAAs) are persistent compounds used in many industrial as well as consumer products. Despite restrictions, these compounds are found at measurable concentrations in samples of human and animal origin. In the present study we examined whether the effects on cell viability of two sulfonated and four carboxylated PFAAs in cultures of cerebellar granule neurons (CGNs), could be associated with deleterious activation of the N-methyl-d-aspartate receptor (NMDA-R). PFAA-induced effects on viability in rat CGNs and unstimulated PC12 cells were examined using the MTT assay. Cells from the PC12 rat pheochromocytoma cell line lack the expression of functional NMDA-Rs and were used to verify lower toxicity of perfluorooctanesulfonic acid (PFOS) in cells not expressing NMDA-Rs. Protective effects of NMDA-R antagonists, and extracellular as well as intracellular Ca2+ chelators were investigated. Cytosolic Ca2+ ([Ca2+]i) was measured using Fura-2. In rat CGNs the effects of the NMDA-R antagonists MK-801, memantine and CPP indicated involvement of the NMDA-R in the decreased viability induced by PFOS and perfluorohexanesulfonic acid (PFHxS). No effects were associated with the four carboxylated PFAAs studied. Further, EGTA and CPP protected against PFOS-induced decreases in cell viability, whereas no protection was afforded by BAPTA-AM. [Ca2+]i significantly increased after exposure to PFOS, and this increase was completely blocked by MK-801. In PC12 cells a higher concentration of PFOS was required to induce equivalent levels of toxicity as compared to in rat CGNs. PFOS-induced toxicity in PC12 cells was not affected by CPP. In conclusion, PFOS at the tested concentrations induces excitotoxicity in rat CGNs, which likely involves influx of extracellular Ca2+ via the NMDA-R. This effect can be blocked by specific NMDA-R antagonists.

Identified lhb-expressing cells from medaka (Oryzias latipes) show similar Ca(2+)-response to all endogenous Gnrh forms, and reveal expression of a novel fourth Gnrh receptor.

We have previously characterized the response to gonadotropin-releasing hormone (Gnrh) 2 in luteinizing hormone (lhb)-expressing cells from green fluorescent protein (Gfp)-transgenic medaka (Oryzias latipes), with regard to changes in the cytosolic Ca(2+) concentration. In the current study we present the corresponding responses to Gnrh1 and Gnrh3. Ca(2+) imaging revealed three response patterns to Gnrh1 and Gnrh3, one monophasic and two types of biphasic patterns. There were few significant differences in the shape of the response patterns between the three Gnrh forms, although the amplitude of the Ca(2+) signal was considerably lower for Gnrh1 and Gnrh3 than for Gnrh2, and the distribution between the two different biphasic patterns differed. The different putative Ca(2+) sources were examined by depleting intracellular Ca(2+) stores with thapsigargin, or preventing influx of extracellular Ca(2+) by either extracellular Ca(2+) depletion or the L-type Ca(2+)-channel blocker verapamil. Both Gnrh1 and 3 relied on Ca(2+) from both intracellular and extracellular sources, with some unexpected differences in the relative contribution. Furthermore, gene expression of Gnrh-receptors (gnrhr) in whole pituitaries was studied during development from juvenile to adult. Only two of the four identified medaka receptors were expressed in the pituitary, gnrhr1b and gnrhr2a, with the newly discovered gnrhr2a showing the highest expression level at all stages as analyzed by quantitative PCR. While both receptors differed in expression level according to developmental stage, only the expression of gnrhr2a showed a clear-cut increase with gonadal maturation. RNA sequencing analysis of FACS-sorted Gfp-positive lhb-cells revealed that both gnrhr1b and gnrhr2a were expressed in lhb-expressing cells, and confirmed the higher expression of gnrhr2a compared to gnrhr1b. These results show that although lhb-expressing gonadotropes in medaka show similar Ca(2+) response patterns to all three endogenous Gnrh forms through the activation of two different receptors, gnrhr1b and gnrhr2a, the differences observed between the Gnrh forms indicate activation of different Ca(2+) signaling pathways.

Electrophysiological differences between fshb- and lhb-expressing gonadotropes in primary culture.

Synthesis and release of FSH and LH are differentially regulated by GnRH, but the mechanisms by which this regulation is achieved are not well understood. Teleost fish are powerful models for studying this differential regulation because they have distinct pituitary cells producing either FSH or LH. By using pituitary cultures from Atlantic cod (Gadus morhua), we were able to investigate and compare the electrophysiological properties of fshb- and lhb-expressing cells, identified by single-cell quantitative PCR after recording. Both cell types fired action potentials spontaneously. The relative number of excitable cells was dependent on reproductive season but varied in opposing directions according to season in the 2 cell types. Excitable and quiescent gonadotropes displayed different ion channel repertoires. The dynamics of outward currents and GnRH-induced membrane responses differed between fshb- and lhb-expressing cells, whereas GnRH-induced cytosolic Ca²âº responses were similar. Expression of Ca²âº-activated K⁺ channels also differed with cell type and showed seasonal variation when measured in whole pituitary. The differential presence of these channels corresponds to the differences observed in membrane response to GnRH. We speculate that differences in ion channel expression levels may be involved in seasonal regulation of hormone secretion as well as the differential response to GnRH in LH- and FSH-producing gonadotropes, through differences in excitability and Ca²âº influx.

Signal transduction involved in GnRH2-stimulation of identified LH-producing gonadotropes from lhb-GFP transgenic medaka (Oryzias latipes).

We have characterized the response to gonadotropin-releasing hormone 2 (GnRH2) in luteinizing hormone producing cells from gfp-transgenic medaka. Teleosts have separate cells producing the two types of gonadotropins, enabling us for the first time to study the intracellular signaling that controls secretion of each gonadotropin separately. Pituitary cell cultures were prepared, and lhb-producing cells were selected by their GFP expression. Cytosolic Ca(2+) imaging revealed three response patterns to GnRH2, one monophasic and two types of biphasic patterns. The Ca(2+) sources were examined by depleting intracellular Ca(2+) stores and preventing influx of extracellular Ca(2+). Both treatments reduced response amplitude, and affected latency and time to peak. Blocking L-type Ca(2+) channels reduced amplitude and time to peak, but did not remove extracellular Ca(2+) contribution. Patch-clamp recordings showed spontaneous action potentials in several cells, and GnRH2 increased the firing frequency. Presence of Ca(2+)-activated K(+) channels was revealed, BK channels being the most prominent.

Trophic structure and community stability in an overfished ecosystem.

Since the collapse of the pelagic fisheries off southwest Africa in the late 1960s, jellyfish biomass has increased and the structure of the Benguelan fish community has shifted, making the bearded goby (Sufflogobius bibarbatus) the new predominant prey species. Despite increased predation pressure and a harsh environment, the gobies are thriving. Here we show that physiological adaptations and antipredator and foraging behaviors underpin the success of these fish. In particular, body-tissue isotope signatures reveal that gobies consume jellyfish and sulphidic diatomaceous mud, transferring "dead-end" resources back into the food chain.