Long-distance mechanism of neurotransmitter recycling mediated by glial network facilitates visual function in Drosophila

Neurons rely on glia to recycle neurotransmitters such as glutamate and histamine for sustained signaling. Both mammalian and insect glia form intercellular gap-junction networks, but their functional significance underlying neurotransmitter recycling is unknown. Using the Drosophila visual system as a genetic model, here we show that a multicellular glial network transports neurotransmitter metabolites between perisynaptic glia and neuronal cell bodies to mediate long-distance recycling of neurotransmitter. In the first visual neuropil (lamina), which contains a multilayer glial network, photoreceptor axons release histamine to hyperpolarize secondary sensory neurons. Subsequently, the released histamine is taken up by perisynaptic epithelial glia and converted into inactive carcinine through conjugation with β-alanine for transport. In contrast to a previous assumption that epithelial glia deliver carcinine directly back to photoreceptor axons for histamine regeneration within the lamina, we detected both carcinine and β-alanine in the fly retina, where they are found in photoreceptor cell bodies and surrounding pigment glial cells. Downregulating Inx2 gap junctions within the laminar glial network causes β-alanine accumulation in retinal pigment cells and impairs carcinine synthesis, leading to reduced histamine levels and photoreceptor synaptic vesicles. Consequently, visual transmission is impaired and the fly is less responsive in a visual alert analysis compared with wild type. Our results suggest that a gap junction-dependent laminar and retinal glial network transports histamine metabolites between perisynaptic glia and photoreceptor cell bodies to mediate a novel, long-distance mechanism of neurotransmitter recycling, highlighting the importance of glial networks in the regulation of neuronal functions.In both vertebrates and insects, glial cells clear neurotransmitters from synaptic clefts (1, 2), thereby increasing the signal-to-noise ratio, enhancing temporal resolution of synaptic transmission, and preventing cross-talk between neighboring neuronal signaling pathways. Once taken into glia, transmitters can be converted into inactive metabolites and sent back to neurons for reuse (3). This glial process of recycling is crucial for neurons to maintain the necessary level of neurotransmitters, such as glutamate and histamine (4, 5), for sustained signal transmission.A variety of transporters for glutamate, dopamine, GABA, glycine, histamine, and their metabolites have been found in both glial and neuronal membranes (6–8). Metabolic enzymes in the recycling process of transmitters, such as glutamate, GABA, and histamine, are also identified (3, 8, 9). However, our knowledge of the trafficking route of inactive transmitter metabolites to neuronal terminals is still lacking. These metabolites may be released from glial processes in close vicinity to neuronal terminals and recycled in an entirely local manner. Alternatively, they may travel within an intercellular glial network and reach a more proximal part of the neuron. Both mammalian and insect glia form intercellular gap-junction networks that allow free diffusion of small molecules (10, 11). The role of these intercellular networks in neurotransmitter recycling is unknown; we are investigating this using the Drosophila visual system as a model.The Drosophila compound eye is composed of hundreds of units named ommatidia. Six peripheral photoreceptors in each ommatidium (12) project axons from the retina to the first layer of visual neuropil, referred to as lamina (5). Within the lamina, photoreceptor axons release histamine upon light stimulation to hyperpolarize projecting large monopolar cells (LMCs) (13). Photoreceptor and LMC axons, and all other neuronal processes in the lamina, are wrapped laterally by epithelial glial cells (14). Whereas the proximal edge of lamina is sealed by marginal glia, the distal edge of laminar neuropil is separated from the retina by four glia layers: two surface (fenestrated and pseudocartridge) glia underneath retina, and distal and proximal satellite glia that wrap cell bodies and initial axon segments of LMCs, respectively (5) (Fig. S1A). Histamine released from photoreceptor axons is removed from the extracellular space by epithelial and probably additional laminar glia (5). In epithelial glia, the N-β-alanyl-biogenic amine synthetase Ebony conjugates histamine to β-alanine to form inactive carcinine for storage and transport (15). Carcinine is then transported back to photoreceptors via an unknown mechanism, where it is hydrolyzed into histamine and β-alanine by a peptidase Tan (9). Compared with de novo synthesis by histidine decarboxylase (HDC) in photoreceptors, the recycling of histamine is more energy efficient and is thus considered to be a dominant pathway in maintaining adequate histamine level (16, 17). Indeed, mutations in both ebony and tan significantly decrease visual histamine level and disrupt fly visual transmission (9).Because Ebony is expressed specifically in epithelial glia in the lamina (18), this glial type was thought to be the only laminar glia mediating histamine recycling until now (Fig. S1A). Given that all laminar glia may form an intercellular network through gap junctions, as evident in a larger fly species Musca (19), it is important to investigate the role of other laminar glia cells in the recycling of histamine. By examining the levels and distributions of histamine, carcinine, and β-alanine in both wild-type and genetically modified flies, we provide evidence that recycling of histamine involves the entire laminar glial network, as well as retinal glia pigment cells.     

MoM cutoffs for variables,an important tool for multivariate analysis and accurate interpretation of preeclampsia risk in high-risk pregnancy at 11–13+6 weeks gestation

Objective: To determine the diagnostic accuracy of preeclampsia (PE) screening test offered in early pregnancy for the prediction of the risks for early-onset (requiring delivery <32 weeks gestation) and late-onset (requiring delivery ≥32 weeks gestation) disease. Methods: In a retrospective study of 615 women with singleton pregnancy, the risk for PE was calculated by the combined effect of multiple variables: serum placental growth factor (PLGF) and pregnancy-associated plasma protein-A (PAPP-A), maternal age, parity, ethnicity, mean arterial pressure (MAP), body mass index (BMI), uterine artery-pulsatility index, and previous history of PE or hypertension (HT). The results of the screening test in three different groups of women were validated by pregnancy outcome: (i) control group – without any history of PE/HT; (ii) history of PE without HT; and (iii) history of HT without PE. The performance of the screening test was evaluated for early- and late-onset PE. Results: The multivariate screening effectively identified cases of PE with >97% specificity. The detection rate (DR) was 93.8% for late-onset PE at a false positive rate (FPR) of 2.3% and 44.4% for early-onset PE at an FPR of 0.0%. The incidence of PE was 7% overall, with 1.52% and 5.43% for early- and late-onset PE, respectively. Conclusion: The study demonstrated 96.6% diagnostic accuracy of the multi-variable screening test to predict the risk of PE in the first trimester. The negative predictive value (>98%) reinforces the utility of cost-effective noninvasive screening test for the early detection of PE.

Abbreviations: PLGF: Placental growth factor; PAPP-A: Pregnancy-associated plasma protein-A; free β-HCG: Free beta-human chorionic gonadotropin; BMI: Body mass index; MAP: Mean arterial blood pressure; Ut-PI: Mean uterine artery pressure (left and right uterine artery)-pulsatility index; MoM: Multiple of median; NICE: National Institute for Health and Clinical Excellence     

Diagnosis of neuroblastoma and ganglioneuroma using Raman spectroscopy

Background

Raman spectroscopy has proven to be useful in studying premalignant and malignant lesions in adults. This is the first report to evaluate Raman spectroscopy in the diagnosis and classification of neuroblastoma in children.

Methods

A biopsy or resection of fresh tissue samples from normal adrenal glands, neuroblastomas, ganglioneuromas, nerve sheath tumors, and pheochromocytoma at our hospital were equally divided between routine histology and spectroscopic studies. At least 12 spectra were collected from different regions of each sample using a Renishaw Raman microscope. Raw spectra were processed to remove noise, florescence, and shot noise, and then analyzed using principle component analysis and discriminant function analysis.

Results

We collected 698 spectra from 16 neuroblastomas, 5 ganglioneuromas, 3 normal adrenal glands, 6 nerve sheath tumors, and 1 pheochromocytoma. Raman spectroscopy differentiated between normal adrenal gland, and neuroblastoma and ganglioneuroma with 100% sensitivity and 100% specificity. It correlated well with the Shimada histologic classification system with 100% sensitivity and 100% specificity. It was also able to differentiate neuroblastoma from nerve sheath tumors and pheochromocytoma with high sensitivity and specificity.

Conclusion

This technique can differentiate neuroblastoma from ganglioneuroma and other tumors. It has a potential as a noninvasive real-time diagnostic tool in classifying pediatric tumors.     

Pre-replication complex organization in the atypical DNA replication cycle of Plasmodium falciparum: characterization of the mini-chromosome maintenance (MCM) complex formation

The overall organization of cell division in Plasmodium is unique compared to that observed in model organisms because DNA replicates more than once per cell cycle at several points of its life cycle. The sequencing of the Plasmodium genome has also revealed the apparent absence of many key components (e.g. Cdt1, DDK and Cdc45) of the eukaryotic cell cycle machinery that are responsible for the formation of the pre-replication complex (pre-RC). We have characterized the Plasmodium falciparum minichromosome maintenance complex (MCM) that plays a key role in the transition of pre-RC to the RC. Similar to other eukaryotes, the Plasmodium genome encodes six MCM subunits. Here, we show that expression levels of at least three of the PfMCM subunits, the homologues of MCM2, MCM6 and MCM7, change during the intraerythrocytic development cycle, peaking in schizont and decreasing in the ring and trophozoite stages. PfMCM2, 6 and 7 subunits interact with each other to form a developmentally regulated complex: these interactions are detectable in rings and schizonts, but not in trophozoites. PfMCM2, 6 and 7 subunits are localized in both cytosolic and nucleosolic fractions during all intraerythrocytic stages of P. falciparum development, with increased nuclear localization in schizonts. Only PfMCM6 is associated with the chromatin fraction at all stages of growth. No phosphorylation of PfMCM2, 6 and 7 was detected, but two as yet unidentified threonine-phosphosphorylated proteins were present in the complex, whose pattern of phosphorylation varied during parasite development.     

Comprehensive Analysis of Recent Biochemical and Biologic Findings Regarding a Newly Discovered Protein-PELP1/MNAR

Estradiol (E2) and estrogen receptor (ER) signaling have been implicated in the development and progression of several cancers. Emerging evidence suggests that the status of ER coregulators in tumor cells plays an important role in hormonal responsiveness and tumor progression. Proline, glutamic acid, and leucine-rich protein-1 (PELP1/MNAR)—a novel ER coactivator that plays an essential role in the ER’s actions and its expression—is deregulated in several hormonal responsive cancers. The precise function of PELP1/MNAR in cancer progression remains unclear, but PELP1 appears to function as a scaffolding protein, coupling ER with several proteins that are implicated in oncogenesis. Emerging evidence suggests that PELP1/MNAR increases E2-mediated cell proliferation and participates in E2-mediated tumorigenesis and metastasis.     

Evaluation of a prime-boost vaccine schedule with distinct adenovirus vectors against malaria in rhesus monkeys

A vaccine that elicits both specific antibodies and IFN-γ-producing T cells is required to protect against pre-erythrocytic malaria. Among the most promising approaches to induce such complex immunity are heterologous prime-boost vaccination regimens, in particular ones containing live viral vector. We have demonstrated previously that adenovectors serotype 35 (Ads35) encoding the circumsporozoite (CS) antigen or liver-stage antigen-1 (LSA-1) are highly effective in improving the T-cell responses induced by immunizations with protein-based vaccines in a heterologous prime-boost schedule. Here we evaluated the potential of a heterologous prime-boost vaccination that combines the Ad35.CS vector with the serologically distinct adenovector Ad5.CS, in rhesus macaques, after establishing the potency in mice. We show that the heterologous Ad35.CS/Ad5.CS prime-boost regimen elicits both antibody responses and robust IFN-γ-producing CD8⁺ T-cell responses against the CS antigen. Analysis of the quality of the antibody responses in rhesus macaques, using indirect immunofluorescence assay (IFA) with Plasmodium falciparum-coated slides, demonstrated that this heterologous prime-boost regimen elicits a high titer of antibodies that are able to bind to P. falciparum sporozoites. Level of the IFA response was superior to the response measured with sera of an adult human population living in endemic malaria region. In conclusion, the combination of Ad35.CS, a vaccine based on a rare serotype adenovirus, with Ad5.CS or possibly another adenovector of a distinct serotype, induces a complex immune response that is required for protection against malaria, and is thus a highly promising approach for pediatric vaccination.