SUMMARY Nutrient amino acid transporters (NATs, subfamily of sodium neurotransmitter symporter family SNF, a.k.a. SLC6) represent a set of phylogenetically and functionally related transport proteins, which perform intracellular absorption of neutral, predominantly essential amino acids. Functions of NATs appear to be critical for the development and survival in organisms. However, mechanisms of specific and synergetic action of various NAT members in the amino acid transport network are virtually unexplored. A new transporter, agNAT8, was cloned from the malaria vector mosquito Anopheles gambiae (SS). Upon heterologous expression in Xenopus oocytes it performs high-capacity, sodium-coupled (2:1)uptake of nutrients with a strong preference for aromatic catechol-branched substrates, especially phenylalanine and its derivatives tyrosine and L-DOPA,but not catecholamines. It represents a previously unknown SNF phenotype, and also appears to be the first sodium-dependent B0 type transporter with a narrow selectivity for essential precursors of catecholamine synthesis pathways. It is strongly and specifically transcribed in absorptive and secretory parts of the larval alimentary canal and specific populations of central and peripheral neurons of visual-, chemo- and mechano-sensory afferents. We have identified a new SNF transporter with previously unknown phenotype and showed its important role in the accumulation and redistribution of aromatic substrates. Our results strongly suggest that agNAT8 is an important, if not the major, provider of an essential catechol group in the synthesis of catecholamines for neurochemical signaling as well as ecdysozoan melanization and sclerotization pathways, which may include cuticle hardening/coloring, wound curing, oogenesis, immune responses and melanization of pathogens.

Damage to the cerebrovascular network is a major contributor to dysfunction in patients suffering from traumatic brain injury (TBI). Vessels are composed of lumen-forming endothelial cells that associate closely with both glial and neuronal units to establish a functional blood–brain barrier (BBB). Under normal physiological conditions, these vascular units play important roles in central nervous system (CNS) homeostasis by delivering oxygen and nutrients while filtering out molecules and cells that could be harmful; however, after TBI this system is disrupted. Here, we describe a novel role for a class of receptors, called dependence receptors, in regulating vessel stability and BBB integrity after CCI injury in mice. Specifically, we identified that EphB3 receptors function as a pro-apoptotic dependence receptor in endothelial cells (ECs) that contributes to increased BBB damage after CCI injury. In the absence of EphB3, we observed increased endothelial cell survival, reduced BBB permeability and enhanced interactions of astrocyte-EC membranes. Interestingly, the brain’s response to CCI injury is to reduce EphB3 levels and its ligand ephrinB3; however, the degree and timing of those reductions limit the protective response of the CNS. We conclude that EphB3 is a negative regulator of cell survival and BBB integrity that undermine tissue repair, and represents a protective therapeutic target for TBI patients.

Alzheimer's disease is characterized mainly by loss of neurons from the septal nucleus. In this study, neurons from the septal nucleus of the embryonic day 16 (E16) rat were grown in culture with a plane of astrocytes from the embryonic rat and in a defined medium in the absence of serum. Neurons were treated with beta-amyloid (Aβ: 0

The senile plaques of Alzheimer's disease contain a high concentration of beta-amyloid (betaA) protein, which may affect the glial population in the septal nucleus, an area of increased risk in AD. BetaA toxicity was measured in septal glia, via a dose-response experiment, by quantifying the effects of three different doses (0.1, 1, and 10 microM) of betaA on cell survival. Astrocytes from embryonic day-16 rats were grown in serum-free media in a single layer culture. Cells were treated on day in vitro (DIV)1 and survival was determined on DIV3 to ascertain which concentration was most toxic. In a separate set of experiments, an attempt was made to protect glial cells from the degenerative effects of betaA, with treatments of growth factors and estrogen. BetaA (10 microM) treatment was administered on DIV1, on DIV2 the cells were treated with estrogen (EST, 10 nM), insulin-like growth factors (IGF1 and IGF2, each 10 ng/ml), basic fibroblast growth factor (bFGF, 5 ng/ml) or nerve growth factor (NGF, 100 ng/ml), and on DIV3 the cells were visualized and quantified by fluorescence microscopy with DAPI (4,6-diamidino-2-phenylindole). In addition to dose-response and glial protection, experiments were also conducted to determine whether toxic effects were due to apoptosis. Our results suggest that the survival of glial populations is significantly affected in all three concentrations (0.1, 1.0, and 10 microM) of betaA. Glial protection was evident in the presence of NGF, for it showed the significantly highest survival rate relative to the betaA treatment alone. Furthermore, toxic effects of betaA appear to be due primarily to apoptosis. Significant reversal of betaA-induced apoptosis was seen with bFGF and IGF1.

Background Traumatic brain injury (TBI) continues to be a major source of death and disability worldwide, and one of the earliest and most profound deficits comes from vascular damage and breakdown of the blood-brain barrier (BBB). Cerebral vascular endothelial cells (cvECs) and endothelial progenitor cells (EPCs) have been shown to play essential roles in vessel repair and BBB stability, although their individual contributions remain poorly defined. New Method We employ TruCount beads with flow cytometry to precisely quantify cvECs, EPCs and peripheral leukocytes in the murine cortex after controlled cortical impact (CCI) injury. Results We found a significant reduction in the number of cvECs at 3 days post-injury (dpi), whereas the EPCs and invading peripheral leukocytes were significantly increased compared with sham controls. Proliferation studies demonstrate that both cvECs and EPCs are undergoing cell expansion in the first week post-injury. Furthermore, analysis of protein expression using mean fluorescent intensity found increases in PECAM-1, VEGFR-2, and VE-Cadherin expression per cell at 3 dpi, which is consistent with western blot analysis. Comparison with Exiting Methods Classic methods of cell analysis, such as histological cell counts, in the traumatic injured brain are labor intensive, time consuming, and potentially biased; whereas flow cytometry provides an efficient, non-biased approach to simultaneously quantify multiple cell types. However, conventional flow cytometry that employs capped events can provide misleading results in CNS injured tissues. Conclusions We demonstrate that TruCount quantification using flow cytometry is a powerful tool for quantifying mature and progenitor endothelial cell changes after TBI.

2 SUMMARY STATEMENTThis article by Laussu et al. describes a role for Eph:ephrin signaling in controlling the identity of neural progenitors in the ventral spinal cord. ABSTRACTEarly specification of progenitors of the ventral spinal cord involves the morphogen Sonic Hedgehog which induces distinct progenitor identities in a dose-dependent manner. Following these initial patterning events, progenitor identities have to be maintained in order to generate appropriate numbers of progeny. Here we provide evidence that communication viaEph:ephrin signaling is required to maintain progenitor identities in the ventral spinal cord.We show that ephrinB2 and ephrinB3 are expressed in restricted progenitor domains in the ventral spinal cord while several Eph receptors are more broadly expressed. Further, we provide evidence that expression of Efnb3 and EphA4 is controlled by Shh. Genetic loss-offunction analyses indicate that expression of ephrinB2 and ephrinB3 is required to control progenitor identities and in vitro experiments reveal that activation of Eph forward signaling in spinal progenitors up-regulates the expression of the identity transcription factor Nkx2.2.Altogether our results indicate that cell-to-cell communication is necessary to control progenitor identity in the ventral spinal cord.

BackgroundIn the vertebrate spinal cord, motor neurons (MN) are generated in stereotypical numbers from a pool of dedicated progenitors (pMN) whose number depends on signals that control their specification but also their proliferation and differentiation rates. Although the initial steps of pMN specification have been extensively studied, how pMN numbers are regulated over time is less well characterized.ResultsHere, we show that ephrinB2 and ephrinB3 are differentially expressed in progenitor domains in the ventral spinal cord with several Eph receptors more broadly expressed. Genetic loss-of-function analyses show that ephrinB2 and ephrinB3 inversely control pMN numbers and that these changes in progenitor numbers correlate with changes in motor neuron numbers. Detailed phenotypic analyses by immunostaining and genetic interaction studies between ephrinB2 and Shh indicate that changes in pMN numbers in ephrin mutants are due to alteration in progenitor identity at late stages of development.ConclusionsAltogether our data reveal that Eph:ephrin signaling is required to control progenitor identities in the ventral spinal cord.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-017-0087-0) contains supplementary material, which is available to authorized users.