Regulation of neuronal excitability by release of proteins from glial cells

Effects of glial cells on electrical isolation and shaping of synaptic transmission between neurons have been extensively studied. Here we present evidence that the release of proteins from astrocytes as well as microglia may regulate voltage-activated Na⁺ currents in neurons, thereby increasing excitability and speed of transmission in neurons kept at distance from each other by specialized glial cells. As a first example, we show that basic fibroblast growth factor and neurotrophin-3, which are released from astrocytes by exposure to thyroid hormone, influence each other to enhance Na⁺ current density in cultured hippocampal neurons. As a second example, we show that the presence of microglia in hippocampal cultures can upregulate Na⁺ current density. The effect can be boosted by lipopolysaccharides, bacterial membrane-derived stimulators of microglial activation. Comparable effects are induced by the exposure of neuron-enriched hippocampal cultures to tumour necrosis factor-α, which is released from stimulated microglia. Taken together, our findings suggest that release of proteins from various types of glial cells can alter neuronal excitability over a time course of several days. This explains changes in neuronal excitability occurring in states of thyroid hormone imbalance and possibly also in seizures triggered by infectious diseases.     

The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins

Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream-acting components like Der1.     

Seeking a spotless mind: extinction, deconsolidation, and erasure of fear memory

Learning to contend with threats in the environment is essential to survival, but dysregulation of memories for traumatic events can lead to disabling psychopathology. Recent years have witnessed an impressive growth in our understanding of the neural systems and synaptic mechanisms underlying emotional memory formation. As a consequence, interest has emerged in developing strategies for suppressing, if not eliminating, fear memories. Here, I review recent work employing sophisticated behavioral, pharmacological, and molecular tools to target fear memories, placing these memories firmly behind the crosshairs of neurobiologically informed interventions.     

Formation and properties of chitosan-cellulose nanocrystal polyelectrolyte-macroion complexes for drug delivery applications

This study examines a novel polyelectrolyte-macroion complex (PMC) between chitosan, a cationic polysaccharide, and cellulose nanocrystals (CNCs), anionic, cylindrical nanoparticles, for potential applications in drug delivery. CNCs were prepared by H(2)SO(4) hydrolysis of wood pulp. The formation of PMCs was monitored by turbidimetric titration. In titrations of a chitosan solution with a CNC suspension, the turbidity reached a plateau, but it had a maximum and then decreased when the direction of titration was reversed. PMC particles were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, dynamic light scattering, and laser Doppler electrophoresis. The particles were composed primarily of CNCs and ranged in size from a few hundred nanometers to several micrometers, depending on the cellulose/chitosan ratio. Particles formed at amino/sulfate group molar ratios >1 were nearly spherical in shape and positively charged, whereas particles formed at ratios <1 had well-defined nonspherical shapes and were negatively charged.     

Against expectation: a short sequence with high signal elucidates cone snail phylogeny

A short (259 nucleotide) conserved intronic sequence (CIS) is surprisingly informative for delineating deep phylogenetic relationships in cone snails. Conus species previously have been assigned to clades based on the evidence from mitochondrial 12S and 16S rRNA gene sequences (1129 bp). Despite their length, these genes lack the phylogenetic information necessary to resolve the relationships among the clades. Here we show that the relationships can be inferred from just 46 sites in the very short CIS sequence (a portion of "intron 9" of the γ-glutamyl carboxylase gene). This is counterintuitive because in short sequences sampling error (noise) often drowns out phylogenetic signal. The intron 9 CIS is rich in synapomorphies that define the divergence patterns among eight clades of worm- and fish-hunting Conus, and it contains almost no homoplasy. Parsimony, maximum likelihood and Bayesian analyses of the combined sequences (mt rRNA+CIS) confirm most of the relationships among 23 Conus sequences. This phylogeny implies that fish-hunting behavior evolved at least twice during the history of Conus-once among New World species and independently in the Indo-Pacific clades.     

Phylogeny of the genus Turris: correlating molecular data with radular anatomy and shell morphology

There are over 10,000 species of venomous marine molluscs, the vast majority of these, which are generally referred to as "turrids", are traditionally assigned to a single family, Turridae (Powell 1966). Here, we provide an initial molecular analysis of the type genus of the family, Turris R?ding, 1798, thought to be among the most well characterized groups in the family. We show that the type genus is not monophyletic. We analyzed specimens conventionally assigned to 9 different Turris species using molecular markers, combined with the shell morphology and radular anatomy whenever feasible. The results suggest that species assigned to the genus Turris, provisionally assigned to two different subgenera are not monophyletic. Five previously described species belong to the subgenus Turris (s.s.) R?ding 1798: Turris babylonia, (Linne, 1758), Turris grandis, (J. E. Gray, 1834), Turris dollyae, (Olivera, 1999), Turris normandavidsoni (Olivera, 1999) and Turris spectabilis (Reeve, 1843). With a change in species designation, Turris assyria (formerly T. babylonia1010) is added to a well-defined clade, which is in turn more closely related to Lophiotoma and Gemmula species than to the other five Turris species. We show that these five species conventionally assigned to Turris do not belong in the same subgenus, and form a clade provisionally designated as AnnulaturrisPowell, 1966: Turris annulata, (Reeve, 1843), Turris undosa, (Lamarck, 1816), Turris cristata, (Vera-Peláez, Vega-Luz, and Lozano-Francisco 2000) Turris cryptorrhaphe (G. B. Sowerby, 1825) and Turris nadaensis (Azuma, 1973). Implications of the molecular phylogenetic results and its correlation with radular morphology are discussed.     

α4β2 nicotinic acetylcholine receptors in the early postnatal mouse superior cervical ganglion

Heteropentameric nicotinic acetylcholine receptors (nAChR) mediate fast synaptic transmission in ganglia of the autonomic nervous system. It is undisputed that α3 and β4 are the predominant subunits in the superior cervical ganglion (SCG); however, reports on the presence of receptors that contain α4 have been controversial. Here, we have searched for the presence of α4-containing nAChRs in the postnatal rat and mouse SCG. We now show by immunoprecipitation combined with radioligand binding that α4-containing receptors constitute about 20% of hetero-oligomeric nAChRs in postnatal Day 3 (P3) mice. However, already by P9, the level of α4 approaches zero. In contrast, the number of α4-containing receptors is close to zero in the rat SCG at all times investigated. Deletion of the β2 subunit by using α5β2-double knockout (KO) mice removes all α4-containing receptors, suggesting that in the postnatal mouse SCG, α4 co-assembles only with β2 but not with β4. α4β2 receptors are, on the other hand, up-regulated in the SCG of P3 α5β4-double KO mice, where they make up about 50% of receptors that bind [(3) H]-epibatidine. Nonetheless, receptors on the surface of SCG neurons from α5β4-double KO mice maintained for one to two days in culture comprise <10% of α4β2 and >90% of α3β2, as determined by patch clamp recordings with α4β2- and α3β2-specific ligands. We propose that in the P3 SCG of wild type mice, α3β4 (±α5) represent about 62% of receptors, whereas 17% are α3β2β4, and 21% are α4β2 (±α5) receptors.     

Towards automated production and drug sensitivity testing using scaffold-free spherical tumor microtissues

Although the relevance of three-dimensional (3-D) culture has been recognized for years and exploited at an academic level, its translation to industrial applications has been slow. The development of reliable high-throughput technologies is clearly a prerequisite for the industrial implementation of 3-D models. In this study the robustness of spherical microtissue production and drug testing in a 96-well hanging-drop multiwell plate format was assessed on a standard 96-well channel robotic platform. Microtissue models derived from six different cell lines were produced and characterized according to their growth profile and morphology displaying high-density tissue-like reformation and growth over at least 15 days. The colon cancer cell line HCT116 was chosen as a model to assess microtissue-based assay reproducibility. Within three individual production batches the size variations of the produced microtissues were below 5%. Reliability of the microtissue-based assay was tested using two reference compounds, staurosporine and chlorambucil. In four independent drug testings the calculated IC(50) values were benchmarked against 2-D multiwell testings displaying similar consistency. The technology presented here for the automated production of a variety of microtissues for efficacy testing in a standard 96-well format will aid the implementation of more organotypic models at an early time point in the drug discovery process.