Nanopores: maltoporin channel as a sensor for maltodextrin and lambda-phage

Background  

To harvest nutrition from the outside bacteria e.g. E. coli developed in the outer cell wall a number of sophisticated channels called porins. One of them, maltoporin, is a passive specific channel for the maltodextrin uptake. This channel was also named LamB as the bacterial virus phage Lambda mis-uses this channel to recognise the bacteria. The first step is a reversible binding followed after a lag phase by DNA injection. To date little is known about the binding capacity and less on the DNA injection mechanism. To elucidate the mechanism and to show the sensitivity of our method we reconstituted maltoporin in planar lipid membranes. Application of an external transmembrane electric field causes an ion current across the channel. Maltoporin channel diameter is around a few Angstroem. At this size the ion current is extremely sensitive to any modification of the channels surface. Protein conformational changes, substrate binding etc will cause fluctuations reflecting the molecular interactions with the channel wall. The recent improvement in ion current fluctuation analysis allows now studying the interaction of solutes with the channel on a single molecular level.     

Complementary encoding of spatial information in hippocampal astrocytes

Calcium dynamics into astrocytes influence the activity of nearby neuronal structures. However, because previous reports show that astrocytic calcium signals largely mirror neighboring neuronal activity, current information coding models neglect astrocytes. Using simultaneous two-photon calcium imaging of astrocytes and neurons in the hippocampus of mice navigating a virtual environment, we demonstrate that astrocytic calcium signals encode (i.e., statistically reflect) spatial information that could not be explained by visual cue information. Calcium events carrying spatial information occurred in topographically organized astrocytic subregions. Importantly, astrocytes encoded spatial information that was complementary and synergistic to that carried by neurons, improving spatial position decoding when astrocytic signals were considered alongside neuronal ones. These results suggest that the complementary place dependence of localized astrocytic calcium signals may regulate clusters of nearby synapses, enabling dynamic, context-dependent variations in population coding within brain circuits.

A combination of functional imaging of astrocytes and neurons in the mouse hippocampus with information theory analysis shows that calcium dynamics in topographically-organized subcellular regions of astrocytes encode information about an animal’s position that is complementary and synergistic to that encoded in the spike output of surrounding neurons.     

Self-Contained Induction of Neurons from Human Embryonic Stem Cells

Background

Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES) cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM). However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins.

Methodology/Principal Findings

We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES) cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM) could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation.

Conclusion/Significance

By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.     

Differential changes in cell morphology,macromolecular composition and membrane protein profiles of neurons and astrocytes in chronic ethanol treated rats

Cellular morphology, macromolecular composition, (DNA, RNA and Protein content) marker enzyme activities for neurons [neuron specific enolase (NSE)] and astrocytes [glutamine synthetase (GS)] and plasma membrane protein profiles in the bulk isolated neurons and astrocytes from control and ethanol treated rats were studied. One month aged Wistar rats were given ethanol as sole drinking fluid for 10 weeks. Scanning electron microscopy revealed a characteristic cell surface smoothening in astrocytes due to ethanol treatment. DNA levels were unaltered, while RNA and Protein contents were decreased in astrocytes and neurons. Further,³H-leucine incorporation into proteins was decreased in neurons and astrocytes derived from ethanol treated rats indicating reduced protein synthesis in neurons and astrocytes. GS activity was affected severely suggesting impairment in astrocytic functions. Plasma membrane protein composition was analyzed by 2-D electrophoresis. The analysis indicated several protein defects in the plasma membranes of neurons and astrocytes, which might be involved in membrane disorder during ethanol challenge.¹²⁵I-Wheat Germ agglutinin binding studies showed three prominent proteins (160, 116 and 97 kDa) in astrocyte membrane fraction suggesting the possible involvement of N-terminal glycoproteins in altered astrocyte morphology during ethanol ingestion. Impairment in the astrocyte cell functions, protein changes in plasma membrane and cellular morphology studies suggest that astrocytes may be more vulnerable than neurons for ethanol effects.     

The expression of hyperpolarization activated cyclic nucleotide gated (HCN) channels in the rat ovary are dependent on the type of cell and the reproductive age of the animal: a laboratory investigation

Background  

Aim of this study was to test the hypothesis that levels of hyperpolarization activated cyclic nucleotide gated channels 1 to 4 (HCN1-4) are linked to the reproductive age of the ovary.     

Trigeminal-Rostral Ventromedial Medulla circuitry is involved in orofacial hyperalgesia contralateral to tissue injury

Background

Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. ?? and ??ENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear.

Results

Here we show that deleting ?? and ??ENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro.

Conclusion

Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.     

Correlations in Ion Channel mRNA in Rhythmically Active Neurons

Background

To what extent do identified neurons from different animals vary in their expression of ion channel genes? In neurons of the same type, is ion channel expression highly variable and/or is there any relationship between ion channel expression that is conserved?

Methodology/Principal Findings

To address these questions we measured ion channel mRNA in large cells (LCs) of the crab cardiac ganglion. We cloned a calcium channel, caco, and a potassium channel, shaker. Using single-cell quantitative PCR, we measured levels of mRNA for these and 6 other different ion channels in cardiac ganglion LCs. Across the population of LCs we measured 3–9 fold ranges of mRNA levels, and we found correlations in the expression of many pairs of conductances

Conclusions/Significance

In previous measurements from the crab stomatogastric ganglion (STG), ion channel expression was variable, but many pairs of channels had correlated expression. However, each STG cell type had a unique combination of ion channel correlations. Our findings from the crab cardiac ganglion are similar, but the correlations in the LCs are different from those in STG neurons, supporting the idea that such correlations could be markers of cell identity or activity.     

Nogo-66 promotes the differentiation of neural progenitors into astroglial lineage cells through mTOR-STAT3 pathway

Background

Neural stem/progenitor cells (NPCs) can differentiate into neurons, astrocytes and oligodendrocytes. NPCs are considered valuable for the cell therapy of injuries in the central nervous system (CNS). However, when NPCs are transplanted into the adult mammalian spinal cord, they mostly differentiate into glial lineage. The same results have been observed for endogenous NPCs during spinal cord injury. However, little is known about the mechanism of such fate decision of NPCs.

Methodology/Principal Findings

In the present study, we have found that myelin protein and Nogo-66 promoted the differentiation of NPCs into glial lineage. NgR and mTOR-Stat3 pathway were involved in this process. Releasing NgR from cell membranes or blocking mTOR-STAT3 could rescue the enhanced glial differentiation by Nogo-66.

Conclusions/Significance

These results revealed a novel function of Nogo-66 in the fate decision of NPCs. This discovery could have profound impact on the understanding of CNS development and could improve the therapy of CNS injuries.     

Network dynamics in nociceptive pathways assessed by the neuronal avalanche model

Background

Imiquimod (IQ) is known as an agonist of Toll-like receptor 7 (TLR7) and is widely used to treat various infectious skin diseases. However, it causes severe itching sensation as its side effect. The precise mechanism of how IQ causes itching sensation is unknown. A recent report suggested a molecular target of IQ as TLR7 expressed in dorsal root ganglion (DRG) neurons. However, we recently proposed a TLR7-independent mechanism, in which the activation of TLR7 is not required for the action of IQ in DRG neurons. To resolve this controversy regarding the involvement of TLR7 and to address the exact molecular identity of itching sensation by IQ, we investigated the possible molecular target of IQ in DRG neurons.

Findings

When IQ was applied to DRG neurons, we observed an increase in action potential (AP) duration and membrane resistance both in wild type and TLR7-deficient mice. Based on these results, we tested whether the treatment of IQ has an effect on the activity of K⁺ channels, K_v1.1 and K_v1.2 (voltage-gated K⁺ channels) and TREK1 and TRAAK (K_2P channels). IQ effectively reduced the currents mediated by both K⁺ channels in a dose-dependent manner, acting as an antagonist at TREK1 and TRAAK and as a partial antagonist at K_v1.1 and K_v1.2.

Conclusions

Our results demonstrate that IQ blocks the voltage-gated K⁺ channels to increase AP duration and K_2P channels to increase membrane resistance, which are critical for the membrane excitability of DRG neurons. Therefore, we propose that IQ enhances the excitability of DRG neurons by blocking multiple potassium channels and causing pruritus.     

Reconstruction and flux analysis of coupling between metabolic pathways of astrocytes and neurons: application to cerebral hypoxia

Background

It is a daunting task to identify all the metabolic pathways of brain energy metabolism and develop a dynamic simulation environment that will cover a time scale ranging from seconds to hours. To simplify this task and make it more practicable, we undertook stoichiometric modeling of brain energy metabolism with the major aim of including the main interacting pathways in and between astrocytes and neurons.

Model

The constructed model includes central metabolism (glycolysis, pentose phosphate pathway, TCA cycle), lipid metabolism, reactive oxygen species (ROS) detoxification, amino acid metabolism (synthesis and catabolism), the well-known glutamate-glutamine cycle, other coupling reactions between astrocytes and neurons, and neurotransmitter metabolism. This is, to our knowledge, the most comprehensive attempt at stoichiometric modeling of brain metabolism to date in terms of its coverage of a wide range of metabolic pathways. We then attempted to model the basal physiological behaviour and hypoxic behaviour of the brain cells where astrocytes and neurons are tightly coupled.

Results

The reconstructed stoichiometric reaction model included 217 reactions (184 internal, 33 exchange) and 216 metabolites (183 internal, 33 external) distributed in and between astrocytes and neurons. Flux balance analysis (FBA) techniques were applied to the reconstructed model to elucidate the underlying cellular principles of neuron-astrocyte coupling. Simulation of resting conditions under the constraints of maximization of glutamate/glutamine/GABA cycle fluxes between the two cell types with subsequent minimization of Euclidean norm of fluxes resulted in a flux distribution in accordance with literature-based findings. As a further validation of our model, the effect of oxygen deprivation (hypoxia) on fluxes was simulated using an FBA-derivative approach, known as minimization of metabolic adjustment (MOMA). The results show the power of the constructed model to simulate disease behaviour on the flux level, and its potential to analyze cellular metabolic behaviour in silico.

Conclusion

The predictive power of the constructed model for the key flux distributions, especially central carbon metabolism and glutamate-glutamine cycle fluxes, and its application to hypoxia is promising. The resultant acceptable predictions strengthen the power of such stoichiometric models in the analysis of mammalian cell metabolism.     

Differential disease resistance response in the barley necrotic mutant nec1

Background  

Although ion fluxes are considered to be an integral part of signal transduction during responses to pathogens, only a few ion channels are known to participate in the plant response to infection. CNGC4 is a disease resistance-related cyclic nucleotide-gated ion channel. Arabidopsis thaliana CNGC4 mutants hlm1 and dnd2 display an impaired hypersensitive response (HR), retarded growth, a constitutively active salicylic acid (SA)-mediated pathogenesis-related response and elevated resistance against bacterial pathogens. Barley CNGC4 shares 67% aa identity with AtCNGC4. The barley mutant nec1 comprising of a frame-shift mutation of CNGC4 displays a necrotic phenotype and constitutively over-expresses PR-1, yet it is not known what effect the nec1 mutation has on barley resistance against different types of pathogens.     

Acidosis-Induced Dysfunction of Cortical GABAergic Neurons through Astrocyte-Related Excitotoxicity

Background

Acidosis impairs cognitions and behaviors presumably by acidification-induced changes in neuronal metabolism. Cortical GABAergic neurons are vulnerable to pathological factors and their injury leads to brain dysfunction. How acidosis induces GABAergic neuron injury remains elusive. As the glia cells and neurons interact each other, we intend to examine the role of the astrocytes in acidosis-induced GABAergic neuron injury.

Results

Experiments were done at GABAergic cells and astrocytes in mouse cortical slices. To identify astrocytic involvement in acidosis-induced impairment, we induced the acidification in single GABAergic neuron by infusing proton intracellularly or in both neurons and astrocytes by using proton extracellularly. Compared the effects of intracellular acidification and extracellular acidification on GABAergic neurons, we found that their active intrinsic properties and synaptic outputs appeared more severely impaired in extracellular acidosis than intracellular acidosis. Meanwhile, extracellular acidosis deteriorated glutamate transporter currents on the astrocytes and upregulated excitatory synaptic transmission on the GABAergic neurons. Moreover, the antagonists of glutamate NMDA-/AMPA-receptors partially reverse extracellular acidosis-induced injury in the GABAergic neurons.

Conclusion

Our studies suggest that acidosis leads to the dysfunction of cortical GABAergic neurons by astrocyte-mediated excitotoxicity, in addition to their metabolic changes as indicated previously.     

A role for thrombospondin-1 deficits in astrocyte-mediated spine and synaptic pathology in Down's syndrome

Background

Down''s syndrome (DS) is the most common genetic cause of mental retardation. Reduced number and aberrant architecture of dendritic spines are common features of DS neuropathology. However, the mechanisms involved in DS spine alterations are not known. In addition to a relevant role in synapse formation and maintenance, astrocytes can regulate spine dynamics by releasing soluble factors or by physical contact with neurons. We have previously shown impaired mitochondrial function in DS astrocytes leading to metabolic alterations in protein processing and secretion. In this study, we investigated whether deficits in astrocyte function contribute to DS spine pathology.

Methodology/Principal Findings

Using a human astrocyte/rat hippocampal neuron coculture, we found that DS astrocytes are directly involved in the development of spine malformations and reduced synaptic density. We also show that thrombospondin 1 (TSP-1), an astrocyte-secreted protein, possesses a potent modulatory effect on spine number and morphology, and that both DS brains and DS astrocytes exhibit marked deficits in TSP-1 protein expression. Depletion of TSP-1 from normal astrocytes resulted in dramatic changes in spine morphology, while restoration of TSP-1 levels prevented DS astrocyte-mediated spine and synaptic alterations. Astrocyte cultures derived from TSP-1 KO mice exhibited similar deficits to support spine formation and structure than DS astrocytes.

Conclusions/Significance

These results indicate that human astrocytes promote spine and synapse formation, identify astrocyte dysfunction as a significant factor of spine and synaptic pathology in the DS brain, and provide a mechanistic rationale for the exploration of TSP-1-based therapies to treat spine and synaptic pathology in DS and other neurological conditions.     

Subthalamic nucleus electrical stimulation modulates calcium activity of nigral astrocytes

Background

The substantia nigra pars reticulata (SNr) is a major output nucleus of the basal ganglia, delivering inhibitory efferents to the relay nuclei of the thalamus. Pathological hyperactivity of SNr neurons is known to be responsible for some motor disorders e.g. in Parkinson''s disease. One way to restore this pathological activity is to electrically stimulate one of the SNr input, the excitatory subthalamic nucleus (STN), which has emerged as an effective treatment for parkinsonian patients. The neuronal network and signal processing of the basal ganglia are well known but, paradoxically, the role of astrocytes in the regulation of SNr activity has never been studied.

Principal Findings

In this work, we developed a rat brain slice model to study the influence of spontaneous and induced excitability of afferent nuclei on SNr astrocytes calcium activity. Astrocytes represent the main cellular population in the SNr and display spontaneous calcium activities in basal conditions. Half of this activity is autonomous (i.e. independent of synaptic activity) while the other half is dependent on spontaneous glutamate and GABA release, probably controlled by the pace-maker activity of the pallido-nigral and subthalamo-nigral loops. Modification of the activity of the loops by STN electrical stimulation disrupted this astrocytic calcium excitability through an increase of glutamate and GABA releases. Astrocytic AMPA, mGlu and GABA_A receptors were involved in this effect.

Significance

Astrocytes are now viewed as active components of neural networks but their role depends on the brain structure concerned. In the SNr, evoked activity prevails and autonomous calcium activity is lower than in the cortex or hippocampus. Our data therefore reflect a specific role of SNr astrocytes in sensing the STN-GPe-SNr loops activity and suggest that SNr astrocytes could potentially feedback on SNr neuronal activity. These findings have major implications given the position of SNr in the basal ganglia network.     

Tetraethylammonium block of water flux in Aquaporin-1 channels expressed in kidney thin limbs of Henle's loop and a kidney-derived cell line.

Background  

Aquaporin-1 (AQP1) channels are constitutively active water channels that allow rapid transmembrane osmotic water flux, and also serve as cyclic-GMP-gated ion channels. Tetraethylammonium chloride (TEA; 0.05 to 10 mM) was shown previously to inhibit the osmotic water permeability of human AQP1 channels expressed in Xenopus oocytes. The purpose of the present study was to determine if TEA blocks osmotic water flux of native AQP1 channels in kidney, and recombinant AQP1 channels expressed in a kidney derived MDCK cell line. We also demonstrate that TEA does not inhibit the cGMP-dependent ionic conductance of AQP1 expressed in oocytes, supporting the idea that water and ion fluxes involve pharmacologically distinct pathways in the AQP1 tetrameric complex.     

Insulin promotes glycogen storage and cell proliferation in primary human astrocytes

Introduction

In the human brain, there are at least as many astrocytes as neurons. Astrocytes are known to modulate neuronal function in several ways. Thus, they may also contribute to cerebral insulin actions. Therefore, we examined whether primary human astrocytes are insulin-responsive and whether their metabolic functions are affected by the hormone.

Methods

Commercially available Normal Human Astrocytes were grown in the recommended medium. Major players in the insulin signaling pathway were detected by real-time RT-PCR and Western blotting. Phosphorylation events were detected by phospho-specific antibodies. Glucose uptake and glycogen synthesis were assessed using radio-labeled glucose. Glycogen content was assessed by histochemistry. Lactate levels were measured enzymatically. Cell proliferation was assessed by WST-1 assay.

Results

We detected expression of key proteins for insulin signaling, such as insulin receptor β-subunit, insulin receptor substrat-1, Akt/protein kinase B and glycogen synthase kinase 3, in human astrocytes. Akt was phosphorylated and PI-3 kinase activity increased following insulin stimulation in a dose-dependent manner. Neither increased glucose uptake nor lactate secretion after insulin stimulation could be evidenced in this cell type. However, we found increased insulin-dependent glucose incorporation into glycogen. Furthermore, cell numbers increased dose-dependently upon insulin treatment.

Discussion

This study demonstrated that human astrocytes are insulin-responsive at the molecular level. We identified glycogen synthesis and cell proliferation as biological responses of insulin signaling in these brain cells. Hence, this cell type may contribute to the effects of insulin in the human brain.     

The evolution of YidC/Oxa/Alb3 family in the three domains of life: a phylogenomic analysis

Background  

YidC/Oxa/Alb3 family includes a group of conserved translocases that are essential for protein insertion into inner membranes of bacteria and mitochondria, and thylakoid membranes of chloroplasts. Because mitochondria and chloroplasts are of bacterial origin, Oxa and Alb3, like many other mitochondrial/chloroplastic proteins, are hypothetically derived from the pre-existing protein (YidC) of bacterial endosymbionts. Here, we test this hypothesis and investigate the evolutionary history of the whole YidC/Oxa/Alb3 family in the three domains of life.     

Behavioural and morphological evidence for the involvement of glial cell activation in delta opioid receptor function: implications for the development of opioid tolerance

The molecular identity and pharmacological properties of mechanically gated ion channels in sensory neurons are poorly understood. We show that FM1-43, a styryl dye used to fluorescently label cell membranes, permeates mechanosensitive ion channels in cultured dorsal root ganglion neurons, resulting in blockade of three previously defined subtypes of mechanically activated currents. Blockade and dye uptake is voltage dependent and regulated by external Ca²⁺. The structurally related larger dye FM3-25 inhibited mechanically activated currents to a lesser degree and did not permeate the channels. In vivo, FMI-43 decreases pain sensitivity in the Randall-Selitto test and increases the withdrawal threshold from von Frey hairs, together suggesting that the channels expressed at the cell body in culture mediate mechanosensation in the intact animal. These data give further insight into the mechanosensitive ion channels expressed by somatosensory neurons and suggest FM dyes are an interesting tool for studying them.     

ZnO nanoparticles modulate the ionic transport and voltage regulation of lysenin nanochannels

Background

The insufficient understanding of unintended biological impacts from nanomaterials (NMs) represents a serious impediment to their use for scientific, technological, and medical applications. While previous studies have focused on understanding nanotoxicity effects mostly resulting from cellular internalization, recent work indicates that NMs may interfere with transmembrane transport mechanisms, hence enabling contributions to nanotoxicity by affecting key biological activities dependent on transmembrane transport. In this line of inquiry, we investigated the effects of charged nanoparticles (NPs) on the transport properties of lysenin, a pore-forming toxin that shares fundamental features with ion channels such as regulation and high transport rate.

Results

The macroscopic conductance of lysenin channels greatly diminished in the presence of cationic ZnO NPs. The inhibitory effects were asymmetrical relative to the direction of the electric field and addition site, suggesting electrostatic interactions between ZnO NPs and a binding site. Similar changes in the macroscopic conductance were observed when lysenin channels were reconstituted in neutral lipid membranes, implicating protein-NP interactions as the major contributor to the reduced transport capabilities. In contrast, no inhibitory effects were observed in the presence of anionic SnO₂ NPs. Additionally, we demonstrate that inhibition of ion transport is not due to the dissolution of ZnO NPs and subsequent interactions of zinc ions with lysenin channels.

Conclusion

We conclude that electrostatic interactions between positively charged ZnO NPs and negative charges within the lysenin channels are responsible for the inhibitory effects on the transport of ions. These interactions point to a potential mechanism of cytotoxicity, which may not require NP internalization.