A functional logic for neurotransmitter corelease in the cholinergic forebrain pathway.

The cholinergic system of the basal forebrain plays an integral part in behaviors ranging from attention to learning, partly by altering the impact of noise in neural populations. The circuit computations underlying cholinergic actions are confounded by recent findings that forebrain cholinergic neurons corelease both acetylcholine (ACh) and GABA. We have identified that corelease of ACh and GABA by cholinergic inputs to the claustrum, a structure implicated in the control of attention, has opposing effects on the electrical activity of claustrum neurons that project to cortical vs. subcortical targets. These actions differentially alter neuronal gain and dynamic range in the two types of neurons. In model networks, the differential effects of ACh and GABA toggle network efficiency and the impact of noise on population dynamics between two different projection subcircuits. Such cholinergic switching between subcircuits provides a potential logic for neurotransmitter corelease in implementing behaviorally relevant computations.

Inducers of the endothelial cell barrier identified through chemogenomic screening in genome-edited hPSC-endothelial cells.

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.

A vertebrate-specific and essential role for osterix in osteogenesis revealed by gene knockout in the teleost medaka.

osterix (osx; sp7) encodes a zinc-finger transcription factor that controls osteoblast differentiation in mammals. Although identified in all vertebrate lineages, its role in non-mammalian bone formation remains elusive. Here, we show that an osx mutation in medaka results in severe bone defects and larval lethality. Pre-osteoblasts fail to differentiate leading to severe intramembranous and perichondral ossification defects. The notochord sheath mineralizes normally, supporting the idea of an osteoblast-independent mechanism for teleost vertebral centra formation. This study establishes a key role for Osx for bone formation in a non-mammalian species, and reveals conserved and non-conserved features in vertebrate bone formation.

Modeling the Effects of Severe Metabolic Disease by Genome Editing of hPSC-Derived Endothelial Cells Reveals an Inflammatory Phenotype.

The kinase AKT2 (PKB) is an important mediator of insulin signaling, for which loss-of-function knockout (KO) mutants lead to early onset diabetes mellitus, and dominant active mutations lead to early development of obesity and endothelial cell (EC) dysfunction. To model EC dysfunction, we used edited human pluripotent stem cells (hPSCs) that carried either a homozygous deletion of AKT2 (AKT2 KO) or a dominant active mutation (AKT2 E17K), which, along with the parental wild type (WT), were differentiated into ECs. Profiling of EC lines indicated an increase in proinflammatory and a reduction in anti-inflammatory fatty acids, an increase in inflammatory chemokines in cell supernatants, increased expression of proinflammatory genes, and increased binding to the EC monolayer in a functional leukocyte adhesion assay for both AKT2 KO and AKT2 E17K. Collectively, these findings suggest that vascular endothelial inflammation that results from dysregulated insulin signaling (homeostasis) may contribute to coronary artery disease, and that either downregulation or upregulation of the insulin pathway may lead to inflammation of endothelial cells. This suggests that the standard of care for patients must be expanded from control of metabolic parameters to include control of inflammation, such that endothelial dysfunction and cardiovascular disorders can ultimately be prevented.

SMN deficiency alters Nrxn2 expression and splicing in zebrafish and mouse models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease affecting lower motor neurons. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, which result in reduced levels of functional SMN protein. Biochemical studies have linked the ubiquitously expressed SMN protein to the assembly of pre-mRNA processing U snRNPs, raising the possibility that aberrant splicing is a major defect in SMA. Accordingly, several transcripts affected upon SMN deficiency have been reported. A second function for SMN in axonal mRNA transport has also been proposed that may likewise contribute to the SMA phenotype. The underlying etiology of SMA, however, is still not fully understood. Here, we have used a combination of genomics and live Ca(2+) imaging to investigate the consequences of SMN deficiency in a zebrafish model of SMA. In a transcriptome analyses of SMN-deficient zebrafish, we identified neurexin2a (nrxn2a) as strongly down-regulated and displaying changes in alternative splicing patterns. Importantly, the knock-down of two distinct nrxn2a isoforms phenocopies SMN-deficient fish and results in a significant reduction of motor axon excitability. Interestingly, we observed altered expression and splicing of Nrxn2 also in motor neurons from the Smn(-/-);SMN2(+/+) mouse model of SMA, suggesting conservation of nrxn2 regulation by SMN in mammals. We propose that SMN deficiency affects splicing and abundance of nrxn2a. This may explain the pre-synaptic defects at neuromuscular endplates in SMA pathophysiology.

Two isoforms of Sister-Of-Mammalian Grainyhead have opposing functions in endothelial cells and in vivo.

OBJECTIVE: Sister-of-Mammalian Grainyhead (SOM) is a member of the Grainyhead family of transcription factors. In humans, 3 isoforms are derived from differential first exon usage and alternative splicing and differ only in their N terminal domain. SOM2, the only variant also present in mouse, induces endothelial cell migration and protects against apoptosis. The functions of the human specific isoforms SOM1 and SOM3 have not yet been investigated. Therefore we wanted to elucidate their functions in endothelial cells. APPROACH AND RESULTS: Overexpression of SOM1 in primary human endothelial cells induced migration, phosphorylation of Akt1 and endothelial nitric oxide synthase, and protected against apoptosis, whereas SOM3 had opposite effects; isoform-specific knockdowns confirmed the disparate effects on apoptosis. After reporter assays demonstrated that both are active transcription factors, microarray analyses revealed that they induce different target genes, which could explain the different cellular effects. Overexpression of SOM3 in zebrafish embryos resulted in increased lethality and severe deformations, whereas SOM1 had no deleterious effect. CONCLUSIONS: Our data demonstrate that the splice variant-derived isoforms SOM1 and SOM3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.

Structure-function analysis of full-length midkine reveals novel residues important for heparin binding and zebrafish embryogenesis.

Midkine is a heparin-binding di-domain growth factor, implicated in many biological processes as diverse as angiogenesis, neurogenesis and tumorigenesis. Elevated midkine levels reflect poor prognosis for many carcinomas, yet the molecular and cellular mechanisms orchestrating its activity remain unclear. At the present time, the individual structures of isolated half domains of human midkine are known and its functionally active C-terminal half domain remains a popular therapeutic target. In the present study, we determined the structure of full-length zebrafish midkine and show that it interacts with fondaparinux (a synthetic highly sulfated pentasaccharide) and natural heparin through a previously uncharacterized, but highly conserved, hinge region. Mutating six consecutive residues in the conserved hinge to glycine strongly abates heparin binding and midkine embryogenic activity. In contrast with previous in vitro studies, we found that the isolated C-terminal half domain is not active in vivo in embryos. Instead, we have demonstrated that the N-terminal half domain is needed to enhance heparin binding and mediate midkine embryogenic activity surprisingly in both heparin-dependent and -independent manners. Our findings provide new insights into the structural features of full-length midkine relevant for embryogenesis, and unravel additional therapeutic routes targeting the N-terminal half domain and conserved hinge.

Pharmacological Enhancement of Adult Hippocampal Neurogenesis Improves Behavioral Pattern Separation in Young and Aged Mice.

BACKGROUND: Impairments in behavioral pattern separation (BPS)-the ability to distinguish between similar contexts or experiences-contribute to memory interference and overgeneralization seen in many neuropsychiatric conditions, including depression, anxiety, PTSD, dementia, and age-related cognitive decline. While BPS relies on the dentate gyrus and is sensitive to changes in adult hippocampal neurogenesis (AHN), its significance as a pharmacological target has not been tested. METHODS: In this study, we applied a human neural stem cell high-throughput screening cascade to identify compounds that increase human neurogenesis. One compound with a favorable profile, RO6871135, was then tested in BPS in mice. RESULTS: Chronic treatment with RO6871135, 7.5 mg/kg increased AHN and improved BPS in a fear discrimination task in both young and aged mice. RO6871135 treatment also lowered innate anxiety-like behavior, which was more apparent in mice exposed to chronic corticosterone. Ablation of AHN by hippocampal irradiation supported a neurogenesis-dependent mechanism for RO6871135-induced improvements in BPS. To identify possible mechanisms of action, in vitro and in vivo kinase inhibition and chemical proteomics assays were performed. These tests indicated that RO6871135 inhibited CDK8, CDK11, CaMK2a, CaMK2b, MAP2K6, and GSK3b. An analog compound also demonstrated high affinity for CDK8, CaMK2a, and GSK3b. CONCLUSIONS: These studies demonstrate a method for empirical identification and preclinical testing of novel neurogenic compounds that can improve BPS, and points to possible novel mechanisms that can be interrogated for the development of new therapies to improve specific endophenotypes such as impaired BPS.

Discovery and optimisation of 1-hydroxyimino-3,3-diphenylpropanes, a new class of orally active GPBAR1 (TGR5) agonists.

A series of non-steroidal GPBAR1 (TGR5) agonists was developed from a hit in a high-throughput screening campaign. Lead identification efforts produced biphenyl-4-carboxylic acid derivative (R)-22, which displayed a robust secretion of PYY after oral administration in a degree that can be correlated with the unbound plasma concentration. Further optimisation work focusing on reduction of the lipophilicity provided the 1-phenylpiperidine-4-carboxylic acid derivative (R)-29 (RO5527239), which showed an improved secretion of PYY and GLP-1, translating into a significant reduction of postprandial blood glucose excursion in an oral glucose tolerance test in DIO mice.

Novel read through agent: ZKN-0013 demonstrates efficacy in APCmin model of familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is a precancerous, colorectal disease characterized by hundreds to thousands of adenomatous polyps caused by mutations in the tumor suppressor gene adenomatous polyposis coli (APC). Approximately 30% of these mutations are premature termination codons (PTC), resulting in the production of a truncated, dysfunctional APC protein. Consequently, the ß-catenin degradation complex fails to form in the cytoplasm, leading to elevated nuclear levels of ß-catenin and unregulated ß-catenin/wnt-pathway signaling. We present in vitro and in vivo data demonstrating that the novel macrolide, ZKN-0013, promotes read through of premature stop codons, leading to functional restoration of full-length APC protein. Human colorectal carcinoma SW403 and SW1417 cells harboring PTC mutations in the APC gene showed reduced levels of nuclear ß-catenin and c-myc upon treatment with ZKN-0013, indicating that the macrolide-mediated read through of premature stop codons produced bioactive APC protein and inhibited the ß-catenin/wnt-pathway. In a mouse model of adenomatous polyposis coli, treatment of APCmin mice with ZKN-0013 caused a significant decrease in intestinal polyps, adenomas, and associated anemia, resulting in increased survival. Immunohistochemistry revealed decreased nuclear ß-catenin staining in the epithelial cells of the polyps in ZKN-0013-treated APCmin mice, confirming the impact on the ß-catenin/wnt-pathway. These results indicate that ZKN-0013 may have therapeutic potential for the treatment of FAP caused by nonsense mutations in the APC gene. KEY MESSAGES: • ZKN-0013 inhibited the growth of human colon carcinoma cells with APC nonsense mutations. • ZKN-0013 promoted read through of premature stop codons in the APC gene. • In APCmin mice, ZKN-0013 treatment reduced intestinal polyps and their progression to adenomas. • ZKN-0013 treatment in APCmin mice resulted in reduced anemia and increased survival.

P2Y12 receptor expression is a critical determinant of functional responsiveness to ATX's MORFO domain.

In the central nervous system, the formation of the myelin sheath and the differentiation of the myelinating cells, namely oligodendrocytes, are regulated by complex signaling networks that involve purinergic receptors and the extracellular matrix. However, the exact nature of the molecular interactions underlying these networks still needs to be defined. In this respect, the data presented here reveal a signaling mechanism that is characterized by an interaction between the purinergic P2Y(12) receptor and the matricellular extracellular matrix protein autotaxin (ATX), also known as ENPP2, phosphodiesterase-Iα/ATX, or lysoPLD. ATX has been previously described by us to mediate intermediate states of oligodendrocyte adhesion and to enable changes in oligodendrocyte morphology that are thought to be crucial for the formation of a fully functional myelin sheath. This functional property of ATX is mediated by ATX's modulator of oligodendrocyte remodeling and focal adhesion organization (MORFO) domain. Here, we show that the expression of the P2Y(12) receptor is necessary for ATX's MORFO domain to exert its effects on differentiating oligodendrocytes. In addition, our data demonstrate that exogenous expression of the P2Y(12) receptor can render cells responsive to the known effects of ATX's MORFO domain, and they identify Rac1 as an intracellular factor mediating the effect of ATX-MORFO-P2Y(12) signaling on the assembly of focal adhesions. Our data further support the idea that a physical interaction between ATX and the P2Y(12) receptor provides the basis for an ATX-MORFO-P2Y(12) signaling axis that is crucial for mediating cellular states of intermediate adhesion and morphological/structural plasticity.

Aluminum Melt Degassing Process Evaluation Depending on the Design and the Degree of the FDU Unit Graphite Rotor Wear.

One of the most important indicators of casting quality is porosity. The formation of pores is largely conditioned by the presence of hydrogen in the batch and subsequently in the melt. The gasification of the melt is the primary factor increasing the porosity of casts. This paper addresses the issue of reducing the melt gasification by using FDU (Foundry Degassing Unit) unit. The gas content in the melt is evaluated by determining the Dichte Index depending on the geometry and the degree of the FDU unit rotor wear. For experiments performed under the operating conditions, three types of graphite rotors with different geometries are used. The extent of melt gasification and the Dichte Index are monitored during the rotor wear, at a rate of 0%, 25%, 50%, 75% and 100% rotor wear. Secondly, the chemical composition of the melt is monitored depending on the design and wear of the rotor. It is proven that the design and the degree of rotor wear do not have significant effect on the chemical composition of the melt and all evaluated samples fell within the prescribed quality in accordance with EN 1706. With regard to the overall comparison of the geometry and wear of individual rotor types, it has been proven that, in terms of efficiency, the individual rotors are mutually equivalent and meet the requirements for melt degassing throughout the service life.

Impact of Rotor Material Wear on the Aluminum Refining Process.

The paper presents the results of tests carried out during the refining of the AlSi9Cu3(Fe) alloy in industrial conditions at the FDU stand. In the tests, three different rotors made of classical graphite, fine-grained graphite and classical graphite with SiC spraying were tested for the degree of wear. A series of tests was conducted for five cases-0% to 100% of consumption every 25%-corresponding to the cycles of the refining process. The number of cycles corresponding to 100% wear of each rotor was determined as 1112. The results of the rotor wear profile for all types of graphite after the assumed cycles are presented. Comparison of CAD models of new rotors and 3D scans of rotors in the final stage of operation revealed material losses during operational tests. The study assessed the efficiency of the rotor in terms of its service life as well as work efficiency. It was estimated on the basis of the calculated values of the Dichte Index (DI) and the density of the samples solidified in the vacuum. The structure of samples before and after refining at various stages of rotor wear is also presented, and the results are discussed.

Using Physical Modeling to Optimize the Aluminium Refining Process.

Concern for the environment and rational management of resources requires the development of recoverable methods of obtaining metallic materials. This also applies to the production of aluminium and its alloys. The quality requirements of the market drive aluminium producers to use effective refining methods, and one of the most commonly used is blowing an inert gas into liquid aluminium via a rotating impeller. The efficiency and cost of this treatment depends largely on the application of the correct ratios between the basic parameters of the process, which are the flow rate of the inert gas, the speed of the rotor and the duration of the process. Determining these ratios in production conditions is expensive and difficult. This article presents the results of research aimed at determining the optimal ratio of the inert gas flow rate to the rotary impeller speed, using physical modeling techniques for the rotor as used in industrial conditions. The tests were carried out for rotary impeller speeds from 150 to 550 rpm and gas flow rates of 12, 17 and 22 dm3/min. The research was carried out on a 1:1 scale physical model, and the results, in the form of visualization of the degree of gas-bubble dispersion, were assessed on the basis of the five typical dispersion patterns. The removal of oxygen from water was carried out analogously to the process of removing hydrogen from aluminium. The curves of the rate of oxygen removal from the model liquid were determined, showing the course of oxygen reduction during refining with the same inert gas flows and rotor speeds mentioned above.

Novel screening cascade identifies MKK4 as key kinase regulating Tau phosphorylation at Ser422.

Phosphorylation of Tau at serine 422 promotes Tau aggregation. The kinase that is responsible for this key phosphorylation event has so far not been identified but could be a potential drug target for Alzheimer's disease. We describe here an assay strategy to identify this kinase. Using a combination of screening a library of 65'000 kinase inhibitors and in vitro inhibitor target profiling of the screening hits using the Ambit kinase platform, MKK4 was identified as playing a key role in Tau-S422 phosphorylation in human neuroblastoma cells.

A neural circuit for excessive feeding driven by environmental context in mice.

Despite notable genetic influences, obesity mainly results from the overconsumption of food, which arises from the interplay of physiological, cognitive and environmental factors. In patients with obesity, eating is determined more by external cues than by internal physiological needs. However, how environmental context drives non-homeostatic feeding is elusive. Here, we identify a population of somatostatin (TNSST) neurons in the mouse hypothalamic tuberal nucleus that are preferentially activated by palatable food. Activation of TNSST neurons enabled a context to drive non-homeostatic feeding in sated mice and required inputs from the subiculum. Pairing a context with palatable food greatly potentiated synaptic transmission between the subiculum and TNSST neurons and drove non-homeostatic feeding that could be selectively suppressed by inhibiting TNSST neurons or the subiculum but not other major orexigenic neurons. These results reveal how palatable food, through a specific hypothalamic circuit, empowers environmental context to drive non-homeostatic feeding.

Neuroscience: A Role for the Claustrum in Drug Reward.

The claustrum is a poorly understood but intriguing part of the brain: a new study has found that it plays an important role in drug reward by providing incentive salience to the location where the drug is administered.

Identification of Mouse Claustral Neuron Types Based on Their Intrinsic Electrical Properties.

Although its dense connections with other brain areas suggests that the claustrum is involved in higher-order brain functions, little is known about the properties of claustrum neurons. Using whole-cell patch clamp recordings in acute brain slices of mice, we characterized the intrinsic electrical properties of more than 300 claustral neurons and used unsupervised clustering of these properties to define distinct cell types. Differences in intrinsic properties permitted separation of interneurons (INs) from projection neurons (PNs). Five subtypes of PNs could be further identified by differences in their adaptation of action potential (AP) frequency and amplitude, as well as their AP firing variability. Injection of retrogradely transported fluorescent beads revealed that PN subtypes differed in their projection targets: one projected solely to subcortical areas while three out of the remaining four targeted cortical areas. INs expressing parvalbumin (PV), somatostatin (SST), or vasoactive intestinal peptide (VIP) formed a heterogenous group. PV-INs were readily distinguishable from VIP-INs and SST-INs, while the latter two were clustered together. To distinguish IN subtypes, an artificial neural network was trained to distinguish the properties of PV-INs, SST-INs, and VIP-INs, as independently identified through their expression of marker proteins. A user-friendly, machine-learning tool that uses intrinsic electrical properties to distinguish these eight different types of claustral cells was developed to facilitate implementation of our classification scheme. Systematic classification of claustrum neurons lays the foundation for future determinations of claustrum circuit function, which will advance our understanding of the role of the claustrum in brain function.

Identification of a combination of transcription factors that synergistically increases endothelial cell barrier resistance.

Endothelial cells (ECs) display remarkable plasticity during development before becoming quiescent and functionally mature. EC maturation is directed by several known transcription factors (TFs), but the specific set of TFs responsible for promoting high-resistance barriers, such as the blood-brain barrier (BBB), have not yet been fully defined. Using expression mRNA data from published studies on ex vivo ECs from the central nervous system (CNS), we predicted TFs that induce high-resistance barrier properties of ECs as in the BBB. We used our previously established method to  generate ECs from human pluripotent stem cells (hPSCs), and then we overexpressed the candidate TFs in hPSC-ECs and measured barrier resistance and integrity using electric cell-substrate impedance sensing, trans-endothelial electrical resistance and FITC-dextran permeability assays. SOX18 and TAL1 were the strongest EC barrier-inducing TFs, upregulating Wnt-related signaling and EC junctional gene expression, respectively, and downregulating EC proliferation-related genes. These TFs were combined with SOX7 and ETS1 that together effectively induced EC barrier resistance, decreased paracellular transport and increased protein expression of tight junctions and induce mRNA expression of several genes involved in the formation of EC barrier and transport. Our data shows identification of a transcriptional network that controls barrier resistance in ECs. Collectively this data may lead to novel approaches for generation of in vitro models of the BBB.