Activity-dependent site-specific changes of glutamate receptor composition in vivo.

The subunit composition of postsynaptic non-NMDA-type glutamate receptors (GluRs) determines the function and trafficking of the receptor. Changes in GluR composition have been implicated in the homeostasis of neuronal excitability and synaptic plasticity underlying learning. Here, we imaged GluRs in vivo during the formation of new postsynaptic densities (PSDs) at Drosophila neuromuscular junctions coexpressing GluRIIA and GluRIIB subunits. GluR composition was independently regulated at directly neighboring PSDs on a submicron scale. Immature PSDs typically had large amounts of GluRIIA and small amounts of GluRIIB. During subsequent PSD maturation, however, the GluRIIA/GluRIIB composition changed and became more balanced. Reducing presynaptic glutamate release increased GluRIIA, but decreased GluRIIB incorporation. Moreover, the maturation of GluR composition correlated in a site-specific manner with the level of Bruchpilot, an active zone protein that is essential for mature glutamate release. Thus, we show that an activity-dependent, site-specific control of GluR composition can contribute to match pre- and postsynaptic assembly.

Opportunities and limitations: A comparative analysis of citizen science and expert recordings for bioacoustic research.

Citizen science is an approach that has become increasingly popular in recent years. Despite this growing popularity, there still is widespread scepticism in the academic world about the validity and quality of data from citizen science projects. And although there might be great potential, citizen science is a rarely used approach in the field of bioacoustics. To better understand the possibilities, but also the limitations, we here evaluated data generated in a citizen science project on nightingale song as a case study. We analysed the quantity and quality of song recordings made in a non-standardized way with a smartphone app by citizen scientists and the standardized recordings made with professional equipment by academic researchers. We made comparisons between the recordings of the two approaches and among the user types of the app to gain insights into the temporal recording patterns, the quantity and quality of the data. To compare the deviation of the acoustic parameters in the recordings with smartphones and professional devices from the original song recordings, we conducted a playback test. Our results showed that depending on the user group, citizen scientists produced many to a lot of recordings of valid quality for further bioacoustic research. Differences between the recordings provided by the citizen and the expert group were mainly caused by the technical quality of the devices used-and to a lesser extent by the citizen scientists themselves. Especially when differences in spectral parameters are to be investigated, our results demonstrate that the use of the same high-quality recording devices and calibrated external microphones would most likely improve data quality. We conclude that many bioacoustic research questions may be carried out with the recordings of citizen scientists. We want to encourage academic researchers to get more involved in participatory projects to harness the potential of citizen science-and to share scientific curiosity and discoveries more directly with society.

A complex system of ligaments and a muscle keep the crystalline lens in place in the eyes of bony fishes (teleosts).

The suspension of the crystalline lens in the eye was studied in 11 species of teleost (bony fish) from 10 families and 7 orders by light and electron microscopy. In all species there were 4-5 ligaments in about the equatorial plane of the eye, in which also the tendon of the retractor lentis muscle attaches to the lens. In two cichlid species two additional ligaments were found running from the mid-posterior surface of the lens to the optic nerve head, where they attach to the falciform process. Lens suspension in teleosts is more complex than previously described and well-suited to firmly keep the heavy spherical lens in position for well-focused vision.

Identification of novel autoantigens via mass spectroscopy-based antibody-mediated identification of autoantigens (MS-AMIDA) using immune thrombocytopenic purpura (ITP) as a model disease.

Immune thrombocytopenic purpura (ITP) is one of the best characterized autoimmune diseases. Autoantibodies (AABs) against platelet antigens are considered as the diagnostic hallmark of ITP, but are detectable in only 50% of patients. We designed and applied a novel proteomic approach termed Mass Spectroscopy-based Antibody-Mediated Identification of Autoantigens (MS-AMIDA) for platelet antigens. Patients were separated into patients with classical AABs [ITP(+)] and patients without AABs [ITP(-)]. Altogether, 181 potential AAGs were found in ITP(+) and 135 AAGs in ITP(-), with 34 and 23 AAGs reproducibly found in two runs of MS-AMIDA. After subtracting identifiers from the controls, 57 AAGs in ITP(+) and 29 AAGs in ITP(+) remained, with 16 AAGs commonly found in ITP(+) and ITP(-) patients. Label-free quantification (LFQ) revealed 15 potential AAGs that are quantitatively stronger in ITP. Dot blot validation was performed on hexokinase 1 (HK1), E1 pyruvate dehydrogenase (E1-PDH), coagulation factor XIII, filamin A (FLNA), non-muscle myosin 9. Eleven patients were found to have anti-HK1 AABs, one patient had anti-E1-PDH AABs, and two patients had anti-FLNA AABs. Most antigens were of intracellular origin with significant association with actin-cytoskeleton and regulation of programmed cell death. In conclusion, novel AAGs for ITP were identified using MS-AMIDA.

Secretome profiling of apheresis platelet supernatants during routine storage via antibody-based microarray.

Platelet storage lesions (PSLs) occur during platelet concentrate (PC) storage. Adverse transfusion reactions (ATRs) have been demonstrated to be more frequent in older PCs and removal of the supernatant prior to transfusion reduces their occurrence. Proteomic profiling of PC supernatants was thus performed to identify proteins associated with PSLs and ATRs. Twenty-four PCs were investigated daily from day 0 to day 9 for platelet pre-activation (PPA), platelet-derived extracellular vesicles (PEVs), and platelet function. Using antibody microarrays, 673 extracellular proteins were analysed in PC supernatants on days 0, 3, 5, 7, and 9. During 5days of storage, PPA and PEVs continuously increased (P<0.0001). Platelet function was observed to remain stable within the first 5days (P=0.1751) and decreased thereafter. Comparison of all time points to day 0 revealed the identification of 136 proteins that were significantly changed in abundance during storage, of which 72 were expressed by platelets. Network analysis identified these proteins to be predominantly associated with exosomes (P=4.61×10-8, n=45 genes) and two clusters with distinct functions were found with one being associated with haemostasis and the other with RNA binding. These findings may provide an explanation for ATRs. SIGNIFICANCE: Changes in platelet concentrate (PC) supernatants during storage have been so far only poorly addressed and high abundant proteins burden the identification of quantitative changes in the secretome. We applied a high-throughput antibody microarray allowing for the sensitive quantification of 673 extracellular factors. PCs account for the highest number of adverse transfusion reactions (ATRs). ATRs have been demonstrated to be more frequent in older PCs and removal of the supernatant prior to transfusion reduces their occurrence. Comprehensive interpretation of the changing proteins in the secretome during platelet storage under blood banking conditions may help to identify mechanisms leading to the occurrence of adverse transfusion reactions.

Gating characteristics control glutamate receptor distribution and trafficking in vivo.

Glutamate-releasing synapses dominate excitatory release in the brain. Mechanisms governing their assembly are of major importance for circuit development and long-term plasticity underlying learning and memory. AMPA/Kainate-type glutamate receptors (GluRs) are tetrameric ligand-gated ion channels that open their ion-conducting pores in response to binding of the neurotransmitter. Changes in subunit composition of postsynaptic GluRs are highly relevant for plasticity and development of glutamatergic synapses [1-4]. To date, posttranslational modifications, mostly operating via the intracellular C-terminal domains (CTDs) of GluRs, are presumed to be the major regulator of trafficking [5]. In recent years, structural and electrophysiological analyses have improved our understanding of GluR gating mechanism [6-11]. However, whether conformational changes subsequent to glutamate binding may per se be able to influence GluR trafficking has remained an unaddressed question. Using a Drosophila system allowing for extended visualization of GluR trafficking in vivo, we here provide evidence that mutations changing the gating behavior alter GluR distribution and trafficking. GluR mutants associated with reduced charge transfer segregated from coexpressed wild-type GluRs on the level of individual postsynaptic densities. Segregation was lost upon blocking of evoked glutamate release. Photobleaching experiments suggested increased mobility of mutants with reduced charge transfer, which accumulated prematurely during early steps of synapse assembly, but failed to further increase their level in accordance with assembly of the presynaptic scaffold. In summary, gating characteristics seem to be a new variable for the understanding of GluR trafficking relevant to both development and plasticity.

Differentially expressed microRNAs in maternal plasma for the noninvasive prenatal diagnosis of Down syndrome (trisomy 21).

OBJECTIVES: Most developmental processes are under the control of small regulatory RNAs called microRNAs (miRNAs). We hypothesize that different fetal developmental processes might be reflected by extracellular miRNAs in maternal plasma and may be utilized as biomarkers for the noninvasive prenatal diagnosis of chromosomal aneuploidies. In this proof-of-concept study, we report on the identification of extracellular miRNAs in maternal plasma of Down syndrome (DS) pregnancies. METHODS: Using high-throughput quantitative PCR (HT-qPCR), 1043 miRNAs were investigated in maternal plasma via comparison of seven DS pregnancies with age and fetal sex matched controls. RESULTS: Six hundred and ninety-five miRNAs were identified. Thirty-six significantly differentially expressed mature miRNAs were identified as potential biomarkers. Hierarchical cluster analysis of these miRNAs resulted in the clear discrimination of DS from euploid pregnancies. Gene targets of the differentially expressed miRNAs were enriched in signaling pathways such as mucin type-O-glycans, ECM-receptor interactions, TGF-beta, and endocytosis, which have been previously associated with DS. CONCLUSIONS: miRNAs are promising and stable biomarkers for a broad range of diseases and may allow a reliable, cost-efficient diagnostic tool for the noninvasive prenatal diagnosis of DS.

Cooperation of Syd-1 with Neurexin synchronizes pre- with postsynaptic assembly.

Synapse formation and maturation requires bidirectional communication across the synaptic cleft. The trans-synaptic Neurexin-Neuroligin complex can bridge this cleft, and severe synapse assembly deficits are found in Drosophila melanogaster neuroligin (Nlg1, dnlg1) and neurexin (Nrx-1, dnrx) mutants. We show that the presynaptic active zone protein Syd-1 interacts with Nrx-1 to control synapse formation at the Drosophila neuromuscular junction. Mutants in Syd-1 (RhoGAP100F, dsyd-1), Nrx-1 and Nlg1 shared active zone cytomatrix defects, which were nonadditive. Syd-1 and Nrx-1 formed a complex in vivo, and Syd-1 was important for synaptic clustering and immobilization of Nrx-1. Consequently, postsynaptic clustering of Nlg1 was affected in Syd-1 mutants, and in vivo glutamate receptor incorporation was changed in Syd-1, Nrx-1 and Nlg1 mutants. Stabilization of nascent Syd-1-Liprin-α (DLiprin-α) clusters, important to initialize active zone formation, was Nlg1 dependent. Thus, cooperation between Syd-1 and Nrx-1-Nlg1 seems to orchestrate early assembly processes between pre- and postsynaptic membranes, promoting avidity of newly forming synaptic scaffolds.

Drosophila neuroligin 1 promotes growth and postsynaptic differentiation at glutamatergic neuromuscular junctions.

Precise apposition of presynaptic and postsynaptic domains is a fundamental property of all neuronal circuits. Experiments in vitro suggest that Neuroligins and Neurexins function as key regulatory proteins in this process. In a genetic screen, we recovered several mutant alleles of Drosophila neuroligin 1 (dnlg1) that cause a severe reduction in bouton numbers at neuromuscular junctions (NMJs). In accord with reduced synapse numbers, these NMJs show reduced synaptic transmission. Moreover, lack of postsynaptic DNlg1 leads to deficits in the accumulation of postsynaptic glutamate receptors, scaffold proteins, and subsynaptic membranes, while increased DNlg1 triggers ectopic postsynaptic differentiation via its cytoplasmic domain. DNlg1 forms discrete clusters adjacent to postsynaptic densities. Formation of these clusters depends on presynaptic Drosophila Neurexin (DNrx). However, DNrx binding is not an absolute requirement for DNlg1 function. Instead, other signaling components are likely involved in DNlg1 transsynaptic functions, with essential interactions organized by the DNlg1 extracellular domain but also by the cytoplasmic domain.