High Content Image Analysis Identifies Novel Regulators of Synaptogenesis in a High-Throughput RNAi Screen of Primary Neurons

The formation of synapses, the specialized points of chemical communication between neurons, is a highly regulated developmental process fundamental to establishing normal brain circuitry. Perturbations of synapse formation and function causally contribute to human developmental and degenerative neuropsychiatric disorders, such as Alzheimer's disease, intellectual disability, and autism spectrum disorders. Many genes controlling synaptogenesis have been identified, but lack of facile experimental systems has made systematic discovery of regulators of synaptogenesis challenging. Thus, we created a high-throughput platform to study excitatory and inhibitory synapse development in primary neuronal cultures and used a lentiviral RNA interference library to identify novel regulators of synapse formation. This methodology is broadly applicable for high-throughput screening of genes and drugs that may rescue or improve synaptic dysfunction associated with cognitive function and neurological disorders.National Institutes of Health (U.S.) (MH095096)National Institutes of Health (U.S.) (R01 GM089652

4D Super-Resolution Microscopy with Conventional Fluorophores and Single Wavelength Excitation in Optically Thick Cells and Tissues

Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample

Task-Related Effects on the Temporal and Spatial Dynamics of Resting-State Functional Connectivity in the Default Network

Recent evidence points to two potentially fundamental aspects of the default network (DN), which have been relatively understudied. One is the temporal nature of the functional interactions among nodes of the network in the resting-state, usually assumed to be static. The second is possible influences of previous brain states on the spatial patterns (i.e., the brain regions involved) of functional connectivity (FC) in the DN at rest. The goal of the current study was to investigate modulations in both the spatial and temporal domains. We compared the resting-state FC of the DN in two runs that were separated by a 45 minute interval containing cognitive task execution. We used partial least squares (PLS), which allowed us to identify FC spatiotemporal patterns in the two runs and to determine differences between them. Our results revealed two primary modes of FC, assessed using a posterior cingulate seed – a robust correlation among DN regions that is stable both spatially and temporally, and a second pattern that is reduced in spatial extent and more variable temporally after cognitive tasks, showing switching between connectivity with certain DN regions and connectivity with other areas, including some task-related regions. Therefore, the DN seems to exhibit two simultaneous FC dynamics at rest. The first is spatially invariant and insensitive to previous brain states, suggesting that the DN maintains some temporally stable functional connections. The second dynamic is more variable and is seen more strongly when the resting-state follows a period of task execution, suggesting an after-effect of the cognitive activity engaged during task that carries over into resting-state periods

Heterogeneous Nuclear Ribonucleoprotein K Interacts with Abi-1 at Postsynaptic Sites and Modulates Dendritic Spine Morphology

BACKGROUND: Abelson-interacting protein 1 (Abi-1) plays an important role for dendritic branching and synapse formation in the central nervous system. It is localized at the postsynaptic density (PSD) and rapidly translocates to the nucleus upon synaptic stimulation. At PSDs Abi-1 is in a complex with several other proteins including WASP/WAVE or cortactin thereby regulating the actin cytoskeleton via the Arp 2/3 complex. PRINCIPAL FINDINGS: We identified heterogeneous nuclear ribonucleoprotein K (hnRNPK), a 65 kDa ssDNA/RNA-binding-protein that is involved in multiple intracellular signaling cascades, as a binding partner of Abi-1 at postsynaptic sites. The interaction with the Abi-1 SH3 domain is mediated by the hnRNPK-interaction (KI) domain. We further show that during brain development, hnRNPK expression becomes more and more restricted to granule cells of the cerebellum and hippocampal neurons where it localizes in the cell nucleus as well as in the spine/dendritic compartment. The downregulation of hnRNPK in cultured hippocampal neurons by RNAi results in an enlarged dendritic tree and a significant increase in filopodia formation. This is accompanied by a decrease in the number of mature synapses. Both effects therefore mimic the neuronal morphology after downregulation of Abi-1 mRNA in neurons. CONCLUSIONS: Our findings demonstrate a novel interplay between hnRNPK and Abi-1 in the nucleus and at synaptic sites and show obvious similarities regarding both protein knockdown phenotypes. This indicates that hnRNPK and Abi-1 act synergistic in a multiprotein complex that regulates the crucial balance between filopodia formation and synaptic maturation in neurons

The Role of the Multiple Banded Antigen of Ureaplasma parvum in Intra-Amniotic Infection: Major Virulence Factor or Decoy?

The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2×104 CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulent-derived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p>0.05). MBA size variants (32–170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1β, IL-6 and IL-8 expression in chorioamnion tissue (p<0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes

Neurexin in Embryonic Drosophila Neuromuscular Junctions

Background: Neurexin is a synaptic cell adhesion protein critical for synapse formation and function. Mutations in neurexin and neurexin-interacting proteins have been implicated in several neurological diseases. Previous studies have described Drosophila neurexin mutant phenotypes in third instar larvae and adults. However, the expression and function of Drosophila neurexin early in synapse development, when neurexin function is thought to be most important, has not been described. Methodology/Principal Findings: We use a variety of techniques, including immunohistochemistry, electron microscopy, in situ hybridization, and electrophysiology, to characterize neurexin expression and phenotypes in embryonic Drosophila neuromuscular junctions (NMJs). Our results surprisingly suggest that neurexin in embryos is present both pre and postsynaptically. Presynaptic neurexin promotes presynaptic active zone formation and neurotransmitter release, but along with postsynaptic neurexin, also suppresses formation of ectopic glutamate receptor clusters. Interestingly, we find that loss of neurexin only affects receptors containing the subunit GluRIIA. Conclusions/Significance: Our study extends previous results and provides important detail regarding the role of neurexin in Drosophila glutamate receptor abundance. The possibility that neurexin is present postsynaptically raises new hypotheses regarding neurexin function in synapses, and our results provide new insights into the role of neurexin i

Resting-State Multi-Spectrum Functional Connectivity Networks for Identification of MCI Patients

In this paper, a high-dimensional pattern classification framework, based on functional associations between brain regions during resting-state, is proposed to accurately identify MCI individuals from subjects who experience normal aging. The proposed technique employs multi-spectrum networks to characterize the complex yet subtle blood oxygenation level dependent (BOLD) signal changes caused by pathological attacks. The utilization of multi-spectrum networks in identifying MCI individuals is motivated by the inherent frequency-specific properties of BOLD spectrum. It is believed that frequency specific information extracted from different spectra may delineate the complex yet subtle variations of BOLD signals more effectively. In the proposed technique, regional mean time series of each region-of-interest (ROI) is band-pass filtered ( Hz) before it is decomposed into five frequency sub-bands. Five connectivity networks are constructed, one from each frequency sub-band. Clustering coefficient of each ROI in relation to the other ROIs are extracted as features for classification. Classification accuracy was evaluated via leave-one-out cross-validation to ensure generalization of performance. The classification accuracy obtained by this approach is 86.5%, which is an increase of at least 18.9% from the conventional full-spectrum methods. A cross-validation estimation of the generalization performance shows an area of 0.863 under the receiver operating characteristic (ROC) curve, indicating good diagnostic power. It was also found that, based on the selected features, portions of the prefrontal cortex, orbitofrontal cortex, temporal lobe, and parietal lobe regions provided the most discriminant information for classification, in line with results reported in previous studies. Analysis on individual frequency sub-bands demonstrated that different sub-bands contribute differently to classification, providing extra evidence regarding frequency-specific distribution of BOLD signals. Our MCI classification framework, which allows accurate early detection of functional brain abnormalities, makes an important positive contribution to the treatment management of potential AD patients

A Glial Variant of the Vesicular Monoamine Transporter Is Required To Store Histamine in the Drosophila Visual System

Unlike other monoamine neurotransmitters, the mechanism by which the brain's histamine content is regulated remains unclear. In mammals, vesicular monoamine transporters (VMATs) are expressed exclusively in neurons and mediate the storage of histamine and other monoamines. We have studied the visual system of Drosophila melanogaster in which histamine is the primary neurotransmitter released from photoreceptor cells. We report here that a novel mRNA splice variant of Drosophila VMAT (DVMAT-B) is expressed not in neurons but rather in a small subset of glia in the lamina of the fly's optic lobe. Histamine contents are reduced by mutation of dVMAT, but can be partially restored by specifically expressing DVMAT-B in glia. Our results suggest a novel role for a monoamine transporter in glia that may be relevant to histamine homeostasis in other systems