72 results found
Karu K, Swanwick RS, Novejarque-Gadea A, et al., 2020, Quantitative proteomic analysis of the central amygdala in neuropathic pain model rats, Journal of Proteome Research, Vol: 19, Pages: 1592-1619, ISSN: 1535-3893
Pain and emotional distress have a reciprocal relation. The amygdala has been implicated in emotional processing. The central nucleus of the amygdala (CeA) receives nociceptive information from the dorsal horn of spinal cord and is responsible for the central plasticity in chronic pain. Neuropathic pain is a type of severe chronic pain and can be strongly influenced by emotional components. Plastic changes in the CeA may play a key role in the development or maintenance or both of neuropathic pain. We studied the expression levels of proteins in the CeA of spinal nerve transection (SNT) model rats. Total tissue lysate proteins were separated by two-dimensional-gel electrophoresis (2D-PAGE). Gels from different time points were compared using Progenesis SameSpot software, and the spots with Fold Change greater than 2 were excised for protein identification by mass spectrometry. We identified more than 50 cytosolic proteins as significantly altered in their expression levels in the CeA of SNT rats, and most of these changes have been validated at mRNA levels by qRT-PCR. We also identified more than 40 membrane proteins as notably up- or down-regulated in the CeA of SNT model rats relative to a control using stable isotope dimethyl labeling nano-LC-MS/MS based proteomics and found that one such protein, doublecortin (DCX), a microtubule-associated protein expressed by neuronal precursor cells during development, is specifically localized in the membrane fraction without changes in total amount of the protein. Immunohistochemistry showed that doublecortin is expressed in processes in the CeA of rats 7 and 21 days after SNT surgery, suggesting that doublecortin is one of the proteins that may contribute to the plastic changes, namely, redevelopment or rewiring of neural networks, in the CeA in the neuropathic pain model. These dysregulated proteins may play roles in reciprocal relationships between pain and psychological distress in the amygdala and contribute to central
Soliman N, Okuse K, Rice A, 2019, VGF, a biomarker and potential target for the treatment of neuropathic pain?, PAIN Reports, Vol: 4, Pages: 1-14, ISSN: 2471-2531
Neuropathic pain remains an area of considerable unmet medical need. A persistentchallenge in the management of neuropathic pain is to target the specific mechanismsleading to a change from normal to abnormal sensory perception while ensuring thatthe defensive pain perception remains intact. Targeting VGF-derived neuropeptidesmay offer this opportunity. VGF was first identified in 1985 and is highly expressedfollowing nerve injury and inflammation in neurons of both the peripheral and centralnervous system. Subsequent studies implicate the vgf gene and its products in painpathways. This narrative review was supported by a systematic search to identify,select and critically appraise all relevant research investigating the role of VGF-derivedneuropeptides in pain pathways. It predominantly focuses on in vivo investigations ofthe role of VGF in the initiation and maintenance of neuropathic pain. VGF expressionlevels are very low under normal physiological conditions and nerve injury results inrapid and robust upregulation, increasing mechanical and thermal hypersensitivity.The identification of the two complement receptors with which VGF-neuropeptidesinteract suggests a novel interplay of neuronal and immune signalling mediators. Theunderstanding of the molecular mechanisms and signalling events by which VGFderivedactive neuropeptides exert their physiological actions is in its infancy. Futurework should aim to improve understanding of the downstream consequences of VGFneuropeptidesthereby providing novel insights into pain mechanisms potentiallyleading to the identification of novel therapeutic targets.
Rizaner N, Onkal R, Fraser SP, et al., 2016, Intracellular calcium oscillations in strongly metastatic human breast and prostate cancer cells: control by voltage-gated sodium channel activity., European Biophysics Journal, Vol: 45, Pages: 735-748, ISSN: 0175-7571
The possible association of intracellular Ca(2+) with metastasis in human cancer cells is poorly understood. We have studied Ca(2+) signaling in human prostate and breast cancer cell lines of strongly versus weakly metastatic potential in a comparative approach. Intracellular free Ca(2+) was measured using a membrane-permeant fluorescent Ca(2+)-indicator dye (Fluo-4 AM) and confocal microscopy. Spontaneous Ca(2+) oscillations were observed in a proportion of strongly metastatic human prostate and breast cancer cells (PC-3M and MDA-MB-231, respectively). In contrast, no such oscillations were observed in weakly/non metastatic LNCaP and MCF-7 cells, although a rise in the resting Ca(2+) level could be induced by applying a high-K(+) solution. Various parameters of the oscillations depended on extracellular Ca(2+) and voltage-gated Na(+) channel activity. Treatment with either tetrodotoxin (a general blocker of voltage-gated Na(+) channels) or ranolazine (a blocker of the persistent component of the channel current) suppressed the Ca(2+) oscillations. It is concluded that the functional voltage-gated Na(+) channel expression in strongly metastatic cancer cells makes a significant contribution to generation of oscillatory intracellular Ca(2+) activity. Possible mechanisms and consequences of the Ca(2+) oscillations are discussed.
Moss PJ, Huang W, Dawes J, et al., 2015, Macrophage-sensory neuronal interaction in HIV-1 gp120-induced neurotoxicity, BRITISH JOURNAL OF ANAESTHESIA, Vol: 114, Pages: 499-508, ISSN: 0007-0912
Okuse K, Ayub M, Swanwick R, et al., 2014, METHODS OF TREATING PAIN BY INHIBITION OF VGF ACTIVITY, WO2013110945
Okuse K, Chen Y-C, Ayub M, et al., 2013, Identification of a receptor for neuropeptide VGF and its role in neuropathic pain, Journal of Biological Chemistry, Vol: n/a, ISSN: 0021-9258
Pristerà A, Baker MD, Okuse K, 2013, Correction: Association between Tetrodotoxin Resistant Channels and Lipid Rafts Regulates Sensory Neuron Excitability., PLoS One, Vol: 8
[This corrects the article on p. e40079 in vol. 7.].
Huang W, Calvo M, Karu K, et al., 2013, A clinically relevant rodent model of HIV antiretroviral drug stavudine induced painful peripheral neuropathy, Pain, Vol: in press
Pristera A, Baker MD, Okuse K, 2012, Association between Tetrodotoxin Resistant Channels and Lipid Rafts Regulates Sensory Neuron Excitability, PLOS ONE, Vol: 7, ISSN: 1932-6203
Antkowiak M, Torres-Mapa ML, McGinty J, et al., 2012, Towards gene therapy based on femtosecond optical transfection, BIOPHOTONICS: PHOTONIC SOLUTIONS FOR BETTER HEALTH CARE III, Vol: 8427, ISSN: 0277-786X
Pristerá A, Okuse K, 2012, Building Excitable Membranes: Lipid Rafts and Multiple Controls on Trafficking of Electrogenic Molecules., Neuroscientist, Vol: 18, Pages: 70-81
Baker MD, Chen Y-C, Shah SU, et al., 2011, In vitro and intrathecal siRNA mediated K(V)1.1 knock-down in primary sensory neurons, MOLECULAR AND CELLULAR NEUROSCIENCE, Vol: 48, Pages: 258-265, ISSN: 1044-7431
Okuse K, Finn A, Pristera A, 2011, Clustering of voltage-gated sodium channel Na(V)1.8 in lipid rafts is essential for action potential propagation in nociceptive unmyelinated axons, 8th EBSA European Biophysics Congress, Publisher: SPRINGER, Pages: 189-189, ISSN: 0175-7571
Swanwick RS, Pristera A, Okuse K, 2010, The trafficking of Na(V)1.8, NEUROSCIENCE LETTERS, Vol: 486, Pages: 78-83, ISSN: 0304-3940
Tsang SW, Shao D, Cheah KS, et al., 2010, Increased basal insulin secretion in Pdzd2-deficient mice, Mol Cell Endocrinol, Vol: 315, Pages: 263-270, ISSN: 1872-8057
Expression of the multi-PDZ protein Pdzd2 (PDZ domain-containing protein 2) is enriched in pancreatic islet beta cells, but not in exocrine or alpha cells, suggesting a role for Pdzd2 in the regulation of pancreatic beta-cell function. To explore the in vivo function of Pdzd2, Pdzd2-deficient mice were generated. Homozygous Pdzd2 mutant mice were viable and their gross morphology appeared normal. Interestingly, Pdzd2-deficient mice showed enhanced glucose tolerance in intraperitoneal glucose tolerance tests and their plasma insulin levels indicated increased basal insulin secretion after fasting. Moreover, insulin release from mutant pancreatic islets was found to be twofold higher than from normal islets. To verify the functional defect in vitro, Pdzd2 was depleted in INS-1E cells using two siRNA duplexes. Pdzd2-depleted INS-1E cells also displayed increased insulin secretion at low concentrations of glucose. Our results provide the first evidence that Pdzd2 is required for normal regulation of basal insulin secretion.
Moss PJ, Okuse K, Rice AS, 2010, HIV-1 GP120-MEDIATED EFFECTS ON NEURITE OUTGROWTH IN CULTURED PRIMARY SENSORY NEURONS, 13th World Congress on Pain
Human immunodeficiency virus-associated sensory neuropathies (HIV-SN) are one of the most frequent neurological complications of HIV infection and its therapy, affecting around 36% of people living with HIV. HIV-SN can result from direct exposure to HIV per se; HIV-associated distal sensory polyneuropathy (HIV-DSP) that is characterised by a ‘die back’ pattern of axonal degeneration of predominantly small diameter neurons and often exhibits as neuropathic pain. Recent research has identified gp120, an HIV viral envelope glycoprotein, to be a key mediator of axonal degeneration and neuronal apoptosis both in vitro and in vivo. However the mechanisms involved and the relative toxicity of its different tropic forms remain unclear. To investigate gp120-mediated neurotoxicity primary rat Dorsal Root Ganglion (DRG) neurons were treated with vehicle or dual tropic gp120MN (20pM-2nM) in Campenot chambers and non-compartmentalised culture systems. These techniques permit investigation of the extent, and temporal profile, of gp120-neurotoxicity both axonally and centrally on the cell body. Neurite density and gp120 localisation were assessed at different time points. Using HCA vision software, neurite density was quantified as the sum of pixels detected for βIII-tubulin staining. Nine images were acquired per coverslip, summed and normalised by dividing by the total number of neuronal cell bodies analysed per coverslip. The mean was calculated from three coverslips per treatment. To differentiate the effects of gp120 on small-unmyelinated nerve fibres and myelinated fibres, βIII-tubulin stained cultures were co-stained for neurofilament-200 (NF200). Neurite outgrowth of NF200 positive and NF200 negative fibres was then assessed. Student’s t-test was employed to determine statistically significantly differences across treatment groups (p<0.05). Gp120MN caused a significant reduction in neurite density in DRG cultures in a dose-dependent manner (2
Ayub M, Chen Y-C, Nagy I, et al., 2010, EFFECTS OF VGF-DERIVED PEPTIDES ON PRIMARY SENSORY NEURONS AND MICROGLIA, 13th World Congress on Pain
VGF (non-acronymic) was first identified as a nerve growth factor (NGF)-inducible gene. It is a 617 amino acid neurosecretory protein, which undergoes endoproteolytic cleavage and the products are released from secretory granules in dense core vesicles upon depolarisation. Several VGF-derived peptides have been characterized and are involved in energy homeostasis, reproductive processes, synaptic plasticity and pain pathways. It has been observed that VGF expression is upregulated in sensory neurons after nerve injury and inflammation and VGF can activate microglia by p38 MAP kinase phosphorylation following injury of peripheral tissue. The identity of all the functional VGF peptides, their receptors and the signalling pathways involved has yet to be elucidated. The aim of this study is to investigate the functional effects of VGF-derived peptides on pain pathways. The fluorescent calcium indicator Fluo-4 was used in primary rat sensory neuron and microglia cultures. The VGF peptides TLQP-21 or LQEQ-19 were applied to the live cells and changes in intracellular calcium concentrations were monitored by fluorescence microscopy. TLQP-21, but not vehicle or LQEQ-19, induced a sharp increase of intracellular calcium concentration, over 100 seconds after application, suggesting a delayed response. These data indicate TLQP-21 may modify microglia activity via a calcium-dependent mechanism. The effects of VGF-derived peptides on alternative cell signalling pathways will also be discussed.
Okuse K, 2010, Clustering of NaV1.8 in lipid rafts on nociceptive unmyelinated axons, 30th European Winter Conference on Brain Research
Maratou K, Wallace VCJ, Hasnie FS, et al., 2009, Comparison of dorsal root ganglion gene expression in rat models of traumatic and HIV-associated neuropathic pain, EUROPEAN JOURNAL OF PAIN, Vol: 13, Pages: 387-398, ISSN: 1090-3801
Pristera A, Okuse K, 2009, Action potential propagation in unmyelinated axons of rat sensory neurons requires association of Nav1.8 channels with the lipid rafts, Society for Neuroscience
We hypothesized that lipid rafts on unmyelinated primary sensory neurons act as a platform on the plasma membrane where Nav1.8 can be clustered to a density that is sufficient to underlie action potentials generation by mimicking the Node of Ranvier in myelinated axons. It is our aim to study the association of Nav1.8 and lipid rafts in unmyelinated sensory neurons and its consequences in action potential propagation in nociceptive sensory neurons.Our data using OptiPrep-gradient ultracentrifugation indicate that Nav1.8 resides both in the non-lipid raft and lipid raft fractions from freshly extracted DRG neurons. Interestingly, Nav1.8 appears to partition into lipid raft fraction upon culture of DRG neurons in the presence of nerve growth factor. It is also confirmed that Nav1.8 localised specifically in the lipid rafts fractions in sciatic nerves. We then used the Cholera Toxin β subunit, anti-caveolin antibody, and anti-Nav1.8 antibody to visualize lipid rafts and Nav1.8 in cultured DRG neurons. The neuronal cell bodies showed a dotted lipid raft staining on the cell membrane, and a diffused cytoplasmatic staining for Nav1.8, without any clear co-localization between them. On the contrary, Nav1.8 showed a striking degree of co-localization with lipid raft markers at the extremity of cell processes and in knobs, at regular intervals, along the neurites. We also investigated the subcellular localization of p11, a factor that has been found to be necessary to functionally expose Nav1.8 onto the membrane. Interestingly, Nav1.8 and p11 share the same pattern of staining and degree of association with lipid rafts in a DRG primary culture. Action potential propagation in unmyelinated sensory axons was visualized by calcium- and voltage-sensitive dyes in response to mechanical stimulation to the axons. Disruption of the lipid rafts in cultured sensory axons by methyl-beta-cyclodextrin caused remarkable reduction in both the conduction velocity and the number of cell
Shao D, Baker MD, Abrahamsen B, et al., 2009, A multi PDZ-domain protein Pdzd2 contributes to functional expression of sensory neuron-specific sodium channel Na(V)1.8, Mol Cell Neurosci, Vol: 42, Pages: 219-225, ISSN: 1095-9327
The voltage-gated sodium channel Na(V)1.8 is expressed exclusively in nociceptive sensory neurons and plays an important role in pain pathways. Na(V)1.8 cannot be functionally expressed in non-neuronal cells even in the presence of beta-subunits. We have previously identified Pdzd2, a multi PDZ-domain protein, as a potential interactor for Na(V)1.8. Here we report that Pdzd2 binds directly to the intracellular loops of Na(V)1.8 and Na(V)1.7. The endogenous Na(V)1.8 current in sensory neurons is inhibited by antisense- and siRNA-mediated downregulation of Pdzd2. However, no marked change in pain behaviours is observed in Pdzd2-decificent mice. This may be due to compensatory upregulation of p11, another regulatory factor for Na(V)1.8, in dorsal root ganglia of Pdzd2-deficient mice. These findings reveal that Pdzd2 and p11 play collaborative roles in regulation of Na(V)1.8 expression in sensory neurons.
Shao D, Okuse K, Djamgoz MB, 2009, Protein-protein interactions involving voltage-gated sodium channels: Post-translational regulation, intracellular trafficking and functional expression, Int J Biochem Cell Biol, Vol: 41, Pages: 1471-1481, ISSN: 1878-5875
Voltage-gated sodium channels (VGSCs), classically known to play a central role in excitability and signalling in nerves and muscles, have also been found to be expressed in a range of 'non-excitable' cells, including lymphocytes, fibroblasts and endothelia. VGSC abnormalities are associated with various diseases including epilepsy, long-QT syndrome 3, Brugada syndrome, sudden infant death syndrome and, more recently, various human cancers. Given their pivotal role in a wide range of physiological and pathophysiological processes, regulation of functional VGSC expression has been the subject of intense study. An emerging theme is post-translational regulation and macro-molecular complexing by protein-protein interactions and intracellular trafficking, leading to changes in functional VGSC expression in plasma membrane. This partially involves endoplasmic reticulum associated degradation and ubiquitin-proteasome system. Several proteins have been shown to associate with VGSCs. Here, we review the interactions involving VGSCs and the following proteins: p11, ankyrin, syntrophin, beta-subunit of VGSC, papin, ERM and Nedd4 proteins. Protein kinases A and C, as well as Ca(2+)-calmodulin dependent kinase II that have also been shown to regulate intracellular trafficking of VGSCs by changing the balance of externalization vs. internalization, and an effort is made to separate these effects from the short-term phosphorylation of mature proteins in plasma membrane. Two further modulatory mechanisms are reciprocal interactions with the cytoskeleton and, late-stage, activity-dependent regulation. Thus, the review gives an updated account of the range of post-translational molecular mechanisms regulating functional VGSC expression. However, many details of VGSC subtype-specific regulation and pathophysiological aspects remain unknown and these are highlighted throughout for completeness.
Okuse K, 2009, Action potential propagation in unmyelinated axons of rat sensory neurons requires clustering of Nav1.8 channels in the lipid rafts, FASEB summer research conference
We hypothesized that lipid rafts on unmyelinated primary sensory neurons act as a platform on the plasma membrane where Nav1.8 can be clustered to a density that is sufficient to underlie action potentials generation by mimicking the Node of Ranvier in myelinated axons. It is our aim to study the association of NaV1.8 and lipid rafts in unmyelinated sensory neurons and its consequences in action potential propagation in nociceptive sensory neurons.Our data using OptiPrep-gradient ultracentrifugation indicate that Nav1.8 resides in the non-lipid raft fractions from freshly extracted DRG neurons. Interestingly, Nav1.8 appears to partition into lipid raft fraction upon culture of DRG neurons in the presence of nerve growth factor. We then used the Cholera Toxin β subunit, anti-caveolin antibody, and anti-Nav1.8 antibody to visualize lipid rafts and NaV1.8 in cultured DRG neurons. The neuronal cell bodies showed a dotted lipid raft staining on the cell membrane, and a diffused cytoplasmatic staining for Nav1.8, without any clear co-localization between them. On the contrary, Nav1.8 showed a striking degree of co-localization with lipid raft markers at the extremity of cell processes and in knobs, at regular intervals, along the neurites. We also investigated the subcellular localization of p11, a factor that has been found to be necessary to functionally expose Nav1.8 onto the membrane. Interestingly, Nav1.8 and p11 share the same pattern of staining and degree of association with lipid rafts in a DRG primary culture. Action potential propagation in unmyelinated sensory axons was visualized by calcium- and voltage-sensitive dyes in response to mechanical stimulation to the axons. Disruption of the lipid rafts in cultured sensory axons by methyl-beta-cyclodextrin caused remarkable reduction in both the conduction velocity and the number of cells responsive to the stimulation.This finding could open a new perspective in the study of subcellular localization mechanism
Huang H-L, Cendan C-M, Roza C, et al., 2008, Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves, MOLECULAR PAIN, Vol: 4
Onkal R, Mattis JH, Fraser SP, et al., 2008, Alternative splicing of Nav1.5: an electrophysiological comparison of 'neonatal' and 'adult' isoforms and critical involvement of a lysine residue, J Cell Physiol, Vol: 216, Pages: 716-726, ISSN: 1097-4652
In developmentally regulated D1:S3 splicing of Nav1.5, there are 31 nucleotide differences between the 5'-exon ('neonatal') and the 3'-exon ('adult') forms, resulting in 7 amino acid differences in D1:S3-S3/S4 linker. In particular, splicing replaces a conserved negative aspartate residue in the 'adult' with a positive lysine. Here, 'neonatal' and 'adult' Nav1.5 alpha-subunit splice variants were stably transfected into EBNA-293 cells and their electrophysiological properties investigated by whole-cell patch-clamp recording. Compared with the 'adult' isoform, the 'neonatal' channel exhibited (1) a depolarized threshold of activation and voltage at which the current peaked; (2) much slower kinetics of activation and inactivation; (3) 50% greater transient charge (Na(+)) influx; (4) a stronger voltage dependence of time to peak; and (5) a slower recovery from inactivation. Tetrodotoxin sensitivity and VGSCbeta1-4 mRNA expression levels did not change. The significance of the charge-reversing aspartate to lysine substitution was investigated by mutating the lysine in the 'neonatal' channel back to aspartate. In this 'neonatal K211D' mutant, the electrophysiological parameters studied strongly shifted back towards the 'adult', that is the lysine residue was primarily responsible for the electrophysiological effects of Nav1.5 D1:S3 splicing. Taken together, these data suggest that the charge reversal in 'neonatal' Nav1.5 would (1) modify the channel kinetics and (2) prolong the resultant current, allowing greater intracellular Na(+) influx. Developmental and pathophysiological consequences of such differences are discussed.
Pristera A, Okuse K, 2008, ASSOCIATION OF NAV1.8 WITH LIPID RAFTS IN RAT DORSAL ROOT GANGLIA NEURON, 12th World Congress of Pain
Aim of Investigation: We hypothesized that lipid rafts on unmyelinated primary sensory neurons act as a platform on the plasma membrane where NaV1.8 can be clustered to a density that is sufficient to underlie action potentials generation by mimicking the Node of Ranvier in myelinated axons. It is our aim to study the association of NaV1.8 and lipid rafts in unmyelinated sensory neurons and its consequences in action potential propagation in nociceptive sensory neurons.Methods: We used OptiPrep-gradient ultracentrifugation in 1% Triton X100 at 4°C followed by SDS-PAGE and Western blotting to identify the lipid raft fraction of rat DRG neurons. We also used immunocytochemistry to identify the localization of lipid rafts and NaV1.8 in cultured DRG neurons and live cell imaging technique using fluorescent protein-tagged NaV1.8.Results: Our data using OptiPrep-gradient ultracentrifugation indicate that NaV1.8 resides in the non-lipid raft fractions from freshly extracted DRG neurons. Interestingly, NaV1.8 appears to partition into lipid raft fraction upon culture of DRG neurons in the presence of nerve growth factor. We then used the Cholera Toxin β subunit, anti-caveolin antibody, and anti-NaV1.8 antibody to visualize lipid rafts and NaV1.8 in cultured DRG neurons. The neuronal cell bodies showed a dotted lipid raft staining on the cell membrane, and a diffused cytoplasmatic staining for NaV1.8, without any clear co-localization between them. On the contrary, NaV1.8 showed a striking degree of co-localization with lipid raft markers at the extremity of cell processes and in knobs, at regular intervals, along the neurites. Live cell imaging using a red fluorescent protein-tagged NaV1.8 expressed in cultured DRG neurons showed a pattern of localization that mirrors the staining of the endogenous NaV1.8 visualized by the antibody. This supports that the punctate localization of NaV1.8 shown by the antibody at lipid rafts is real and not artifacts of fixation/anti
Okuse K, 2007, Pain signalling pathways: From cytokines to ion channels, INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY, Vol: 39, Pages: 490-496, ISSN: 1357-2725
Okuse K, Chaplan SR, Wood JN, 2007, Regulation of expression of the sensory neuron-specific sodium channel SNS in inflammatory and neuropathic pain, Society for Neuroscience
Foulkes T, Nassar MA, Lane T, et al., 2006, Deletion of annexin 2 light chain p11 in nociceptors causes deficits in somatosensory coding and pain behavior, JOURNAL OF NEUROSCIENCE, Vol: 26, Pages: 10499-10507, ISSN: 0270-6474
Fang X, Djouhri L, McMullan S, et al., 2006, Intense isolectin-B-4 binding in rat dorsal root ganglion neurons distinguishes C-fiber nociceptors with broad action potentials and high Nav1.9 expression, JOURNAL OF NEUROSCIENCE, Vol: 26, Pages: 7281-7292, ISSN: 0270-6474
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