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Genetic and clinical association studies have identified disrupted in schizophrenia 1 (DISC1) as a candidate risk gene for major mental illness. DISC1 is interrupted by a balanced chr(1;11) translocation in a Scottish family in which the translocation predisposes to psychiatric disorders. We investigate the consequences of DISC1 interruption in human neural cells using TALENs or CRISPR-Cas9 to target the DISC1 locus. We show that disruption of DISC1 near the site of the translocation results in decreased DISC1 protein levels because of nonsense-mediated decay of long splice variants. This results in an increased level of canonical Wnt signaling in neural progenitor cells and altered expression of fate markers such as Foxg1 and Tbr2. These gene expression changes are rescued by antagonizing Wnt signaling in a critical developmental window, supporting the hypothesis that DISC1-dependent suppression of basal Wnt signaling influences the distribution of cell types generated during cortical development.

Microglia, the resident immune cells of the central nervous system (CNS), are responsible for maintaining homeostasis in the brain by clearing debris and are suggested to be inefficient in Alzheimer's Disease (AD), a progressive neurodegenerative disorder for which there is no disease-modifying drug. Besides pathological approaches, unbiased evidence from genome-wide association studies (GWAS) and gene network analysis implicate genes expressed in microglia that reduce phagocytic ability as susceptibility genes for AD. Thus, a central feature toward AD therapy is to increase the microglial phagocytic activities while maintaining synaptic integrity. Here, we developed a robust unbiased high content screening assay to identify potential therapeutics which can reduce the amyloid-beta (Aβ1–42) load by increasing microglial uptake ability. Our screen identified the small-molecule GW5074, an inhibitor of c-RAF, a serine/threonine kinase, which significantly increased the Aβ1–42 clearance activities in human monocyte-derived microglia-like (MDMi) cells, a microglia culture model that recapitulates many genetic and phenotypic aspects of human microglia. Notably, GW5074 was previously reported to be neuroprotective for cerebellar granule cells and cortical neurons. We found that GW5074 significantly increased the expression of key AD-associated microglial molecules known to modulate phagocytosis: TYROBP, SIRPβ1, and TREM2. Our results demonstrated that GW5074 is a potential therapeutic for AD, by targeting microglia. [ABSTRACT FROM AUTHOR]

Synthetic oxytocin (sOT) is widely used during labor, yet little is known about its effects on fetal brain development despite evidence that it reaches the fetal circulation. Here, we tested the hypothesis that sOT would affect early neurodevelopment by investigating its effects on neural progenitor cells (NPC) from embryonic day 14 rat pups. NPCs expressed the oxytocin receptor (OXTR), which was downregulated by 45% upon prolonged treatment with sOT. Next, we examined the effects of sOT on NPC death, apoptosis, proliferation, and differentiation using antibodies to NeuN (neurons), Olig2 (oligodendrocytes), and GFAP (astrocytes). Treated NPCs were analysed with unbiased high-throughput immunocytochemistry. Neither 6 nor 24 h exposure to 100 pM or 100 nM sOT had an effect on viability as assessed by PI or CC-3 immunocytochemistry. Similarly, sOT had negligible effect on NPC proliferation, except that the overall rate of NPC proliferation was higher in the 24 h compared to the 6 h group regardless of sOT exposure. The most significant finding was that sOT exposure caused NPCs to select a predominantly neuronal lineage, along with a concomitant decrease in glial cells. Collectively, our data suggest that perinatal exposure to sOT can have neurodevelopmental consequences for the fetus, and support the need for in vivo anatomical and behavioral studies in offspring exposed to sOT in utero. [ABSTRACT FROM AUTHOR]

Neurocognitive dysfunction is common in survivors of intensive care. Prolonged sedation has been implicated but the mechanisms are unclear. Neurogenesis continues into adulthood and is implicated in learning. The neural progenitor cells (NPC) that drive neurogenesis have receptors for the major classes of sedatives used clinically, suggesting that interruption of neurogenesis may partly contribute to cognitive decline in ICU survivors. Using an in vitro system, we tested the hypothesis that prolonged exposure to propofol concentration- and duration-dependently kills or markedly decreases the proliferation of NPCs. NPCs isolated from embryonic day 14 Sprague-Dawley rat pups were exposed to 0, 2.5, or 5.0 μg/mL of propofol, concentrations consistent with deep clinical anesthesia, for either 4 or 24 hours. Cells were assayed for cell death and proliferation either immediately following propofol exposure or 24 hours later. NPC death and apoptosis were measured by propidium iodine staining and cleaved caspase-3 immunocytochemistry, respectively, while proliferation was measured by EdU incorporation. Staurosporine (1μM for 6h) was used as a positive control for cell death. Cells were analyzed with unbiased high-throughput immunocytochemistry. There was no cell death at either concentration of propofol or duration of exposure. Neither concentration of propofol impaired NPC proliferation when exposure lasted 4 h, but when exposure lasted 24 h, propofol had an anti-proliferative effect at both concentrations (P < 0.0001, propofol vs. control). However, this effect was transient; proliferation returned to baseline 24 h after discontinuation of propofol (P = 0.37, propofol vs. control). The transient but reversible suppression of NPC proliferation, absence of cytotoxicity, and negligible effect on the neural stem cell pool pool suggest that propofol, even in concentrations used for clinical anesthesia, has limited impact on neural progenitor cell biology. [ABSTRACT FROM AUTHOR]

Background: The ability to grow a uniform cell type from the adult central nervous system (CNS) is valuable for developing cell therapies and new strategies for drug discovery. The adult mammalian brain is a source of neural stem cells (NSC) found in both neurogenic and non-neurogenic zones but difficulties in culturing these hinders their use as research tools [1,2,3,4,5,6]. Methodology/Principal Findings: Here we show that NSCs can be efficiently grown in adherent cell cultures when angiogenic signals are included in the medium. These signals include both anti-angiogenic factors (the soluble form of the Notch receptor ligand, Dll4) and pro-angiogenic factors (the Tie-2 receptor ligand, Angiopoietin 2). These treatments support the self renewal state of cultured NSCs and expression of the transcription factor Hes3, which also identifies the cancer stem cell population in human tumors. In an organotypic slice model, angiogenic factors maintain vascular structure and increase the density of dopamine neuron processes. Conclusions/Significance: We demonstrate new properties of adult NSCs and a method to generate efficient adult NSC cultures from various central nervous system areas. These findings will help establish cellular models relevant to cancer and regeneration. [ABSTRACT FROM AUTHOR]

Many Gram-negative bacteria utilize specialized secretion systems to inject proteins (effectors) directly into host cells. Little is known regarding how bacteria ensure that only small subsets of the thousands of proteins they encode are recognized as substrates of the secretion systems, limiting their identification through bioinformatic analyses. Many of these proteins require chaperones to direct their secretion. Here, using the newly described protein interaction platform assay, we demonstrate that type 3 secretion system class IB chaperones from one bacterium directly bind their own effectors as well as those from other species. In addition, we observe that expression of class IB homologs from seven species, including pathogens and endosymbionts, mediate the translocation of effectors from Shigella directly into host cells, demonstrating that class IB chaperones are often functionally interchangeable. Notably, class IB chaperones bind numerous effectors. However, as previously proposed, they are not promiscuous; rather they recognize a defined sequence that we designate the conserved chaperone-binding domain (CCBD) sequence [(LMIF)\(_{1}\)XXX(IV)\(_{5}\)XX(IV)\(_{8}\)X(N)\(_{10}\)]. This sequence is the first defined amino acid sequence to be identified for any interspecies bacterial secretion system, i.e., a system that delivers proteins directly into eukaryotic cells. This sequence provides a new means to identify substrates of type III secretion systems. Indeed, using a pattern search algorithm for the CCBD sequence, we have identified the first two probable effectors from an endosymbiont, Sodalis glossinidius.

Recent work shows that major developmental and clinical processes such as central nervous system regeneration and carcinogenesis involve stem cells (SCs) in the brain. In spite of this importance, the requirements of these SCs and their differentiated offspring (neurons, astrocytes, and oligodendrocytes) for survival and proper function are little understood. In vivo, the SCs themselves interact with their environment. This "SC niche" may be complex because it likely includes cells of the vascular and immune systems. The ability to maintain (1) and differentiate (1 -4) central nervous system (CNS) SCs in tissue culture where they can be pharmacologically or genetically (5) manipulated provides a powerful starting point for understanding their behavior. We present detailed information on the methods that permit CNS SCs to differentiate into functional neurons in tissue culture. Important aspects of the culture systems include (1) homogeneity, so that the input and output of a manipulation is known to involve the SC itself; (2) growth in monolayer to visualize and study individual SCs and their offspring; and (3) the use of fully defined culture components to exclude unknown factors from the culture. These conditions support the differentiation of functional, electrically active neurons. These methods allow cell growth and differentiation from normal adult and diseased tissue derived from both animal models and clinical samples. Ultimate validation of such a system comes from accurate prediction of in vivo effects, and the methods we present for CNS SC culture have also successfully predicted regenerative responses in the injured adult nervous system.