The pro-inflammatory oxidant hypochlorous acid induces Bax-dependent mitochondrial permeabilisation and cell death through AIF-/EndoG-dependent pathways

At sites of chronic inflammation, such as in the inflamed rheumatoid joint, activated neutrophils release hydrogen peroxide (H2O2) and the enzyme myeloperoxidase to catalyse the formation of hypochlorous acid (HOCl). 3-chlorotyrosine, a marker of HOCl in vivo, has been observed in synovial fluid proteins from rheumatoid arthritis patients. However the mechanisms of HOCl-induced cytotxicity are unknown. We determined the molecular mechanisms by which HOCl induced cell death in human mesenchymal progenitor cells (MPCs) differentiated into a chondrocytic phenotype as a model of human cartilage cells and show that HOCl induced rapid Bax conformational change, mitochondrial permeability and release of intra-mitochondrial pro-apoptotic proteins which resulted in nuclear translocation of AIF and EndoG. siRNA-mediated knockdown of Bax substantially prevented mitochondrial permeability, release of intra-mitochondrial pro-apoptotic proteins. Cell death was inhibited by siRNA-mediated knockdown of Bax, AIF or EndoG. Although we observed several biochemical markers of apoptosis, caspase activation was not detected either by western blotting, fluorescence activity assays or by using caspase inhibitors to inhibit cell death. This was further supported by findings that (1) in vitro exposure of recombinant human caspases to HOCl caused significant inhibition of caspase activity and (2) the addition of HOCl to staurosporine-treated MPCs inhibited the activity of cellular caspases. Our results show for the first time that HOCl induced Bax-dependent mitochondrial permeability which led to cell death without caspase activity by processes involving AIF/EndoG-dependent pathways. Our study provides a novel insight into the potential mechanisms of cell death in the inflamed human joint. (c) 2006 Elsevier Inc. All rights reserved.

Activation of pro-survival Akt and ERK1/2 signalling pathways underlie the anti-apoptotic effects of flavanones in cortical neurons

There is growing interest in the potential beneficial effects of flavonoids in the aging and diseased brain. We have investigated the potential of the flavanone hesperetin and two of its metabolites, hesperetin-7-O-beta-D-glucuronide and 5-nitro-hesperetin, to inhibit oxidative stress-induced neuronal apoptosis. Exposure of cortical neurons to hydrogen peroxide led to the activation of apoptosis signal-regulating kinase 1 via its de-phosphorylation at Ser963, the phosphorylation of c-jun N-terminal kinase and c-Jun (Ser73) and the activation of caspase 3 and caspase 9. Whilst hesperetin glucuronide failed to exert protection, both hesperetin and 5-nitro-hesperetin were effective at preventing neuronal apoptosis via a mechanism involving the activation/phosphorylation of both Akt/protein kinase B and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Protection against oxidative injury and the activation of Akt and ERK1/2 followed a bell-shaped response and was most apparent at 100 nmol/L concentrations. The activation of ERK1/2 and Akt by flavanones led to the inhibition of the pro-apoptotic proteins, apoptosis signal-regulating kinase 1, by phosphorylation at Ser83 and Bad, by phosphorylation at both Ser136 and Ser112 and to the inhibition of peroxide-induced caspase 9 and caspase 3 activation. Thus, flavanones may protect neurons against oxidative insults via the modulation of neuronal apoptotic machinery.

Flavonoids and brain health: multiple effects underpinned by common mechanisms

The neuroprotective actions of dietary flavonoids involve a number of effects within the brain, including a potential to protect neurons against injury induced by neurotoxins, an ability to suppress neuroinflammation, and the potential to promote memory, learning and cognitive function. This multiplicity of effects appears to be underpinned by two processes. Firstly, they interact with important neuronal signalling cascades leading to an inhibition of apoptosis triggered by neurotoxic species and to a promotion of neuronal survival and differentiation. These interactions include selective actions on a number of protein kinase and lipid kinase signalling cascades, most notably the PI3K/Akt and MAP kinase pathways which regulate pro-survival transcription factors and gene expression. Secondly, they induce peripheral and cerebral vascular blood flow in a manner which may lead to the induction of angiogenesis, and new nerve cell growth in the hippocampus. Therefore, the consumption of flavonoid-rich foods, such as berries and cocoa, throughout life holds a potential to limit the neurodegeneration associated with a variety of neurological disorders and to prevent or reverse normal or abnormal deteriorations in cognitive performance.

The impact of flavonoids on memory: physiological and molecular considerations

Emerging evidence suggests that a group of dietary-derived phytochemicals known as flavonoids are able to induce improvements in memory acquisition, consolidation, storage and retrieval. These low molecular weight polyphenols are widespread in the human diet, are absorbed to only a limited degree and localise in the brain at low concentration. However, they have been found to be highly effective in reversing age-related declines in memory via their ability to interact with the cellular and molecular architecture of the brain responsible for memory. These interactions include an ability to activate signalling pathways, critical in controlling synaptic plasticity, and a potential to induce vascular effects capable of causing new nerve cell growth in the hippocampus. Their ability to activate the extracellular signal-regulated kinase (ERK1/2) and the protein kinase B (PKB/Akt) signalling pathways, leading to the activation of the cAMP response element-binding protein (CREB), a transcription factor responsible for increasing the expression of a number of neurotrophins important in de. ning memory, will be discussed. How these effects lead to improvements in memory through induction of synapse growth and connectivity, increases in dendritic spine density and the functional integration of old and new neurons will be illustrated. The overall goal of this critical review is to emphasize future areas of investigation as well as to highlight these dietary agents as promising candidates for the design of memory-enhancing drugs with relevance to normal and pathological brain ageing (161 references).

The interactions of flavonoids within neuronal signalling pathways

Emerging evidence suggests that dietary phytochemicals, in particular flavonoids, may exert beneficial effects in the central nervous system by protecting neurons against stress-induced injury, by suppressing neuroinflammation and by promoting neurocognitive performance, through changes in synaptic plasticity. It is likely that flavonoids exert such effects in neurons, through selective actions on different components within a number of protein kinase and lipid kinase signalling cascades, such as phosphatidylinositol-3 kinase (PI3K)/Akt, protein kinase C and mitogen-activated protein kinase. This review details the potential inhibitory or stimulatory actions of flavonoids within these pathways, and describes how such interactions are likely to affect cellular function through changes in the activation state of target molecules and/or by modulating gene expression. Although, precise sites of action are presently unknown, their abilities to: (1) bind to ATP binding sites on enzymes and receptors; (2) modulate the activity of kinases directly; (3) affect the function of important phosphatases; (4) preserve neuronal Ca2+ homeostasis; and (5) modulate signalling cascades lying downstream of kinases, are explored. Future research directions are outlined in relation to their precise site(s) of action within the signalling pathways and the sequence of events that allow them to regulate neuronal function in the central nervous system.

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin- stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca2+ mobilisation. in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1 alpha (D26A) (MIP-1 alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCLS), MIP-1 beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin -stimulated CAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCLS, CCL8 and CCL13 were able to stimulate Ca2+ mobilisation. through CCRS, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca2+ responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca2+ mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca2+ mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCRS. (C) 2007 Elsevier Inc. All rights reserved.

Invasive neural prosthesis for neural signal detection and nerve stimulation

This paper specifically examines the implantation of a microelectrode array into the median nerve of the left arm of a healthy male volunteer. The objective was to establish a bi-directional link between the human nervous system and a computer, via a unique interface module. This is the first time that such a device has been used with a healthy human. The aim of the study was to assess the efficacy, compatibility, and long term operability of the neural implant in allowing the subject to perceive feedback stimulation and for neural activity to be detected and processed such that the subject could interact with remote technologies. A case study demonstrating real-time control of an instrumented prosthetic hand by means of the bi-directional link is given. The implantation did not result in infection, and scanning electron microscope images of the implant post extraction have not indicated significant rejection of the implant by the body. No perceivable loss of hand sensation or motion control was experienced by the subject while the implant was in place, and further testing of the subject following the removal of the implant has not indicated any measurable long term defects. The implant was extracted after 96 days. Copyright © 2004 John Wiley & Sons, Ltd.

Gene transcription in Daphnia magna: effects of acute exposure to a carbamate insecticide and an acetanilide herbicide.

Daphnia magna is a key invertebrate in the freshwater environment and is used widely as a model in ecotoxicological measurements and risk assessment. Understanding the genomic responses of D. magna to chemical challenges will be of value to regulatory authorities worldwide. Here we exposed D. magna to the insecticide methomyl and the herbicide propanil to compare phenotypic effects with changes in mRNA expression levels. Both pesticides are found in drainage ditches and surface water bodies standing adjacent to crops. Methomyl, a carbamate insecticide widely used in agriculture, inhibits acetylcholinesterase, a key enzyme in nerve transmission. Propanil, an acetanilide herbicide, is used to control grass and broad-leaf weeds. The phenotypic effects of single doses of each chemical were evaluated using a standard immobilisation assay. Immobilisation was linked to global mRNA expression levels using the previously estimated 48h-EC(1)s, followed by hybridization to a cDNA microarray with more than 13,000 redundant cDNA clones representing >5000 unique genes. Following exposure to methomyl and propanil, differential expression was found for 624 and 551 cDNAs, respectively (one-way ANOVA with Bonferroni correction, Ppathways. Pesticide-specific toxic responses were found but there is little evidence for transcriptional responses purely restricted to genes associated with the pesticide target site or mechanism of toxicity.

The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

Pathways to inflated responsibility beliefs in adolescent obsessive-compulsive disorder: a preliminary investigation

Background: An inflated sense of responsibility is characteristic of obsessive-compulsive disorder (OCD). No previous studies have investigated its origins. Five potential pathways to inflated responsibility beliefs have been proposed; these are tested in this study. Method: A novel measure, the Origins Questionnaire for Adolescents (OQA), was developed to assess experiences on these five pathways. Reliability of the OQA was investigated. The experiences on the five pathways to inflated responsibility beliefs of sixteen adolescents with a history of OCD were compared to sixteen adolescents with no history of OCD. Parents also reported on adolescents’ experiences on the five pathways. Results: Inter-rater reliability was high. The internal consistency of the subscales were only partly satisfactory. The groups differed on one pathway; the clinical group reported a higher sense of responsibility for significant incidents with a negative outcome prior to onset of OCD. Conclusions: An inflated sense of responsibility, in combination with the occurrence of specific incidents, might act as a vulnerability factor for development of OCD. Future research should consider how to measure the subtle effects of experiences of responsibility over the course of development.

Crosstalk with insulin and dependence on PI3K/Akt/mTOR rather than MAPK pathways in upregulation of basal growth following long-term oestrogen deprivation in three human breast cancer cell lines

Background: MCF-7, T-47-D, ZR-75-1 human breast cancer cell lines are dependent on oestrogen for growth but can adapt to grow during long-term oestrogen deprivation. This serves as a model for identification of therapeutic targets in endocrine-resistant breast cancer. Methods: An overlooked complication of this model is that it involves more than non-addition of oestrogen, and inadequate attention has been given to separating molecular events associated with each of the culture manipulations. Results: Insulin and oestradiol were shown to protect MCF-7 cells against upregulation of basal growth, demonstrating a crosstalk in the growth adaptation process. Increased phosphorylation of p44/42MAPK and c-Raf reflected removal of insulin from the medium and proliferation of all three cell lines was inhibited to a lesser extent by PD98059 and U0126 following long-term oestrogen/insulin withdrawal, demonstrating a reduced dependence on the MAPK pathway. By contrast, long-term oestrogen/insulin deprivation did not alter levels of phosphorylated Akt and did not alter the dose-response of growth inhibition with LY294002 in any of the three cell lines. The IGF1R inhibitor picropodophyllin inhibited growth of all MCF-7 cells but only in the long-term oestrogen/insulin-deprived cells was this paralleled by reduction in phosphorylated p70S6K, a downstream target of mTOR. Long-term oestrogen/insulin-deprived MCF-7 cells had higher levels of phosphorylated p70S6K and developed increased sensitivity to growth inhibition by rapamycin. Conclusions: The greater sensitivity to growth inhibition by rapamycin in all three cell lines following long-term oestrogen/insulin deprivation suggests rapamycin-based therapies might be more effective in breast cancers with acquired oestrogen resistance. Keywords Akt, breast cancer cells, endocrine resistance, insulin, MAPK, MCF-7 cells, mTOR, oestrogen, oestrogen-deprived, PI3K, picropodophyllin, rapamycin, T-47-D cells, ZR-75-1 cells

Inhibition of synaptic transmission and G protein modulation by synthetic CaV2.2 Ca2+ channel peptides

Abstract: Modulation of presynaptic voltage-dependent Ca+ channels is a major means of controlling neurotransmitter release. The CaV 2.2 Ca2+ channel subunit contains several inhibitory interaction sites for Gβγ subunits, including the amino terminal (NT) and I–II loop. The NT and I–II loop have also been proposed to undergo a G protein-gated inhibitory interaction, whilst the NT itself has also been proposed to suppress CaV 2 channel activity. Here, we investigate the effects of an amino terminal (CaV 2.2[45–55]) ‘NT peptide’ and a I–II loop alpha interaction domain (CaV 2.2[377–393]) ‘AID peptide’ on synaptic transmission, Ca2+ channel activity and G protein modulation in superior cervical ganglion neurones (SCGNs). Presynaptic injection of NT or AID peptide into SCGN synapses inhibited synaptic transmission and also attenuated noradrenaline-induced G protein modulation. In isolated SCGNs, NT and AID peptides reduced whole-cell Ca2+ current amplitude, modified voltage dependence of Ca2+ channel activation and attenuated noradrenaline-induced G protein modulation. Co-application of NT and AID peptide negated inhibitory actions. Together, these data favour direct peptide interaction with presynaptic Ca2+ channels, with effects on current amplitude and gating representing likely mechanisms responsible for inhibition of synaptic transmission. Mutations to residues reported as determinants of Ca2+ channel function within the NT peptide negated inhibitory effects on synaptic transmission, Ca2+ current amplitude and gating and G protein modulation. A mutation within the proposed QXXER motif for G protein modulation did not abolish inhibitory effects of the AID peptide. This study suggests that the CaV 2.2 amino terminal and I–II loop contribute molecular determinants for Ca2+ channel function; the data favour a direct interaction of peptides with Ca2+ channels to inhibit synaptic transmission and attenuate G protein modulation. Non-technical summary: Nerve cells (neurones) in the body communicate with each other by releasing chemicals (neurotransmitters) which act on proteins called receptors. An important group of receptors (called G protein coupled receptors, GPCRs) regulate the release of neurotransmitters by an action on the ion channels that let calcium into the cell. Here, we show for the first time that small peptides based on specific regions of calcium ion channels involved in GPCR signalling can themselves inhibit nerve cell communication. We show that these peptides act directly on calcium channels to make them more difficult to open and thus reduce calcium influx into native neurones. These peptides also reduce GPCR-mediated signalling. This work is important in increasing our knowledge about modulation of the calcium ion channel protein; such knowledge may help in the development of drugs to prevent signalling in pathways such as those involved in pain perception.

Prosthesis grasp reflex via peripheral nerve control: an in vitro study

Here we present an economical and versatile platform for developing motor control and sensory feedback of a prosthetic hand via in vitro mammalian peripheral nerve activity. In this study, closed-loop control of the grasp function of the prosthetic hand was achieved by stimulation of a peripheral nerve preparation in response to slip sensor data from a robotic hand, forming a rudimentary reflex action. The single degree of freedom grasp was triggered by single unit activity from motor and sensory fibers as a result of stimulation. The work presented here provides a novel, reproducible, economic, and robust platform for experimenting with neural control of prosthetic devices before attempting in vivo implementation.

New insights on regulation of LMTK2, a membrane kinase integrating pathways central to neurodegeneration

In a short communication in this issue (Manser et al. 2012), Christopher Miller’s group at the Institute of Psychiatry, King’s College London present an elegant and convincing set of experiments using molecular techniques to show that a brain-enriched membrane-associated protein kinase, lemur tyrosine kinase-2 (LMTK2), is directly phosphorylated by the cyclin-dependent kinase-5/p35 and this event is sufficient for LMTK2 to phosphorylate an abundant protein phosphatase, PP1C. LMTK2 has been little studied to date and, despite its name, is a kinase which phosphorylates serine or threonine residues of protein substrates. The paper adds to the evidence that this enzyme is a potentially important mediator positioned to integrate a number of intracellular signalling pathways relevant to neurodegeneration.