Role of 1-Deoxysphingolipids in docetaxel neurotoxicity.

A major dose-limiting side effect of docetaxel chemotherapy is peripheral neuropathy. Patients' symptoms include pain, numbness, tingling and burning sensations, and motor weakness in the extremities. The molecular mechanism is currently not understood, and there are no treatments available. Previously, we have shown an association between neuropathy symptoms of patients treated with paclitaxel and the plasma levels of neurotoxic sphingolipids, the 1-deoxysphingolipids (1-deoxySL) (Kramer et al, FASEB J, 2015). 1-DeoxySL are produced when the first enzyme of the sphingolipid biosynthetic pathway, serine palmitoyltransferase (SPT), uses L-alanine as a substrate instead of its canonical amino acid substrate, L-serine. In the current investigation, we tested whether 1-deoxySL accumulate in the nervous system following systemic docetaxel treatment in mice. In dorsal root ganglia (DRG), we observed that docetaxel (45 mg/kg cumulative dose) significantly elevated the levels of 1-deoxySL and L-serine-derived ceramides, but not sphingosine-1-phosphate (S1P). S1P is a bioactive sphingolipid and a ligand for specific G-protein-coupled receptors. In the sciatic nerve, docetaxel decreased 1-deoxySL and ceramides. Moreover, we show that in primary DRG cultures, 1-deoxysphingosine produced neurite swellings that could be reversed with S1P. Our results demonstrate that docetaxel chemotherapy up-regulates sphingolipid metabolism in sensory neurons, leading to the accumulation of neurotoxic 1-deoxySL. We suggest that the neurotoxic effects of 1-deoxySL on axons can be reversed with S1P.

Methylglyoxal and a spinal TRPA1-AC1-Epac cascade facilitate pain in the db/db mouse model of type 2 diabetes.

Painful diabetic neuropathy (PDN) is a devastating neurological complication of diabetes. Methylglyoxal (MG) is a reactive metabolite whose elevation in the plasma corresponds to PDN in patients and pain-like behavior in rodent models of type 1 and type 2 diabetes. Here, we addressed the MG-related spinal mechanisms of PDN in type 2 diabetes using db/db mice, an established model of type 2 diabetes, and intrathecal injection of MG in conventional C57BL/6J mice. Administration of either a MG scavenger (GERP10) or a vector overexpressing glyoxalase 1, the catabolic enzyme for MG, attenuated heat hypersensitivity in db/db mice. In C57BL/6J mice, intrathecal administration of MG produced signs of both evoked (heat and mechanical hypersensitivity) and affective (conditioned place avoidance) pain. MG-induced Ca2+ mobilization in lamina II dorsal horn neurons of C57BL/6J mice was exacerbated in db/db, suggestive of MG-evoked central sensitization. Pharmacological and/or genetic inhibition of transient receptor potential ankyrin subtype 1 (TRPA1), adenylyl cyclase type 1 (AC1), protein kinase A (PKA), or exchange protein directly activated by cyclic adenosine monophosphate (Epac) blocked MG-evoked hypersensitivity in C57BL/6J mice. Similarly, intrathecal administration of GERP10, or inhibitors of TRPA1 (HC030031), AC1 (NB001), or Epac (HJC-0197) attenuated hypersensitivity in db/db mice. We conclude that MG and sensitization of a spinal TRPA1-AC1-Epac signaling cascade facilitate PDN in db/db mice. Our results warrant clinical investigation of MG scavengers, glyoxalase inducers, and spinally-directed pharmacological inhibitors of a MG-TRPA1-AC1-Epac pathway for the treatment of PDN in type 2 diabetes.

Complement 3a receptor in dorsal horn microglia mediates pronociceptive neuropeptide signaling.

The complement 3a receptor (C3aR1) participates in microglial signaling under pathological conditions and was recently shown to be activated by the neuropeptide TLQP-21. We previously demonstrated that TLQP-21 elicits hyperalgesia and contributes to nerve injury-induced hypersensitivity through an unknown mechanism in the spinal cord. Here we determined that this mechanism requires C3aR1 and that microglia are the cellular target for TLQP-21. We propose a novel neuroimmune signaling pathway involving TLQP-21-induced activation of microglial C3aR1 that then contributes to spinal neuroplasticity and neuropathic pain. This unique dual-ligand activation of C3aR1 by a neuropeptide (TLQP-21) and an immune mediator (C3a) represents a potential broad-spectrum mechanism throughout the CNS for integration of neuroimmune crosstalk at the molecular level.

Spinal 12-lipoxygenase-derived hepoxilin A3 contributes to inflammatory hyperalgesia via activation of TRPV1 and TRPA1 receptors.

Peripheral inflammation initiates changes in spinal nociceptive processing leading to hyperalgesia. Previously, we demonstrated that among 102 lipid species detected by LC-MS/MS analysis in rat spinal cord, the most notable increases that occur after intraplantar carrageenan are metabolites of 12-lipoxygenases (12-LOX), particularly hepoxilins (HXA(3) and HXB(3)). Thus, we examined involvement of spinal LOX enzymes in inflammatory hyperalgesia. In the current work, we found that intrathecal (IT) delivery of the LOX inhibitor nordihydroguaiaretic acid prevented the carrageenan-evoked increase in spinal HXB(3) at doses that attenuated the associated hyperalgesia. Furthermore, IT delivery of inhibitors targeting 12-LOX (CDC, Baicalein), but not 5-LOX (Zileuton) dose-dependently attenuated tactile allodynia. Similarly, IT delivery of 12-LOX metabolites of arachidonic acid 12(S)-HpETE, 12(S)-HETE, HXA(3), or HXB(3) evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA(3) produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA(3) correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA(3) triggered sustained mobilization of calcium in cells stably overexpressing TRPV1 or TRPA1 receptors and in acutely dissociated rodent sensory neurons. Constitutive deletion or antagonists of TRPV1 (AMG9810) or TRPA1 (HC030031) attenuated this action. Furthermore, pretreatment with antihyperalgesic doses of AMG9810 or HC030031 reduced spinal HXA(3)-evoked allodynia. These data indicate that spinal HXA(3) is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.

SHIP1 therapeutic target enablement: Identification and evaluation of inhibitors for the treatment of late-onset Alzheimer's disease.

INTRODUCTION: The risk of developing Alzheimer's disease is associated with genes involved in microglial function. Inositol polyphosphate-5-phosphatase (INPP5D), which encodes Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1), is a risk gene expressed in microglia. Because SHIP1 binds receptor immunoreceptor tyrosine-based inhibitory motifs (ITIMs), competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P2, it is a negative regulator of microglia function. Validated inhibitors are needed to evaluate SHIP1 as a potential therapeutic target. METHODS: We identified inhibitors and screened the enzymatic domain of SHIP1. A protein construct containing two domains was used to evaluate enzyme inhibitor potency and selectivity versus SHIP2. Inhibitors were tested against a construct containing all ordered domains of the human and mouse proteins. A cellular thermal shift assay (CETSA) provided evidence of target engagement in cells. Phospho-AKT levels provided further evidence of on-target pharmacology. A high-content imaging assay was used to study the pharmacology of SHIP1 inhibition while monitoring cell health. Physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties were evaluated to select a compound suitable for in vivo studies. RESULTS: SHIP1 inhibitors displayed a remarkable array of activities and cellular pharmacology. Inhibitory potency was dependent on the protein construct used to assess enzymatic activity. Some inhibitors failed to engage the target in cells. Inhibitors that were active in the CETSA consistently destabilized the protein and reduced pAKT levels. Many SHIP1 inhibitors were cytotoxic either at high concentration due to cell stress or they potently induced cell death depending on the compound and cell type. One compound activated microglia, inducing phagocytosis at concentrations that did not result in significant cell death. A pharmacokinetic study demonstrated brain exposures in mice upon oral administration. DISCUSSION: 3-((2,4-Dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine activated primary mouse microglia and demonstrated exposures in mouse brain upon oral dosing. Although this compound is our recommended chemical probe for investigating the pharmacology of SHIP1 inhibition at this time, further optimization is required for clinical studies. Highlights: Cellular thermal shift assay (CETSA) and signaling (pAKT) assays were developed to provide evidence of src homology 2 (SH2) domain-contaning inositol phosphatase 1 (SHIP1) target engagement and on-target activity in cellular assays.A phenotypic high-content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was developed to explore cellular pharmacology and monitor cell health.SHIP1 inhibitors demonstrate a wide range of activity and cellular pharmacology, and many reported inhibitors are cytotoxic.The chemical probe 3-((2,4-dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine is recommended to explore SHIP1 pharmacology.

Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons.

BACKGROUND: Central sensitization in the spinal cord requires glutamate receptor activation and intracellular Ca2+ mobilization. We used Fura-2 AM bulk loading of mouse slices together with wide-field Ca2+ imaging to measure glutamate-evoked increases in extracellular Ca2+ to test the hypotheses that: 1. Exogenous application of glutamate causes Ca2+ mobilization in a preponderance of dorsal horn neurons within spinal cord slices taken from adult mice; 2. Glutamate-evoked Ca2+ mobilization is associated with spontaneous and/or evoked action potentials; 3. Glutamate acts at glutamate receptor subtypes to evoked Ca2+ transients; and 4. The magnitude of glutamate-evoked Ca2+ responses increases in the setting of peripheral neuropathic pain. RESULTS: Bath-applied glutamate robustly increased [Ca2+]i in 14.4 ± 2.6 cells per dorsal horn within a 440 x 330 um field-of-view, with an average time-to-peak of 27 s and decay of 112 s. Repeated application produced sequential responses of similar magnitude, indicating the absence of sensitization, desensitization or tachyphylaxis. Ca2+ transients were glutamate concentration-dependent with a Kd = 0.64 mM. Ca2+ responses predominantly occurred on neurons since: 1) Over 95% of glutamate-responsive cells did not label with the astrocyte marker, SR-101; 2) 62% of fura-2 AM loaded cells exhibited spontaneous action potentials; 3) 75% of cells that responded to locally-applied glutamate with a rise in [Ca2+]i also showed a significant increase in AP frequency upon a subsequent glutamate exposure; 4) In experiments using simultaneous on-cell recordings and Ca2+ imaging, glutamate elicited a Ca2+ response and an increase in AP frequency. AMPA/kainate (CNQX)- and AMPA (GYKI 52466)-selective receptor antagonists significantly attenuated glutamate-evoked increases in [Ca2+]i, while NMDA (AP-5), kainate (UBP-301) and class I mGluRs (AIDA) did not. Compared to sham controls, peripheral nerve injury significantly decreased mechanical paw withdrawal threshold and increased glutamate-evoked Ca2+ signals. CONCLUSIONS: Bulk-loading fura-2 AM into spinal cord slices is a successful means for determining glutamate-evoked Ca2+ mobilization in naïve adult dorsal horn neurons. AMPA receptors mediate the majority of these responses. Peripheral neuropathic injury potentiates Ca2+ signaling in dorsal horn.

Hydroxy-epoxide and keto-epoxide derivatives of linoleic acid activate trigeminal neurons.

Endogenous lipid mediators are proposed to contribute to headache and facial pain by activating trigeminal neurons (TN). We recently identified 11-hydroxy-epoxide- and 11-keto-epoxide derivatives of linoleic acid (LA) that are present in human skin and plasma and potentially contribute to nociception. Here we expand upon initial findings by examining the effects of 11-hydroxy- and 11-keto-epoxide-LA derivatives on TN activation in comparison to LA, the LA derivative [9-hydroxy-octadecadienoic acid (9-HODE)] and prostaglandin E2 (PGE2). 11-hydroxy- and 11-keto-epoxide-LA derivatives elicited Ca2+ transients in TN subpopulations. The proportion of neurons responding to test compounds (5 µM, 5 min) ranged from 16.2 ± 3.8 cells (11 K-9,10E-LA) to 34.1 ± 2.4 cells (11H-12,13E-LA). LA and 9-HODE (5 µM, 5 min) elicited responses in 11.6 ± 3.1% and 9.7 ± 3.4% of neurons, respectively. 11H-12,13E-LA, 11K-12,13E-LA, and 11H-9,10E-LA produced Ca2+ responses in significantly higher proportions of neurons compared to either LA or 9-HODE (F (6, 36) = 5.12, P = 0.0007). 11H-12,13E-LA and 11H-9,10E-LA increased proportions of responsive neurons in a concentration-dependent fashion, similar to PGE2. Most sensitive neurons responded to additional algesic agents (32.9% to capsaicin, 40.1% to PGE2, 58.0% to AITC), however 20.6% did not respond to any other agent. In summary, 11-hydroxy-epoxide derivatives of LA increase trigeminal neuron excitability, suggesting a potential role in headache or facial pain.

Opioid receptors inhibit the spinal AMPA receptor Ca2+ permeability that mediates latent pain sensitization.

Acute inflammation induces sensitization of nociceptive neurons and triggers the accumulation of calcium permeable (CP) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) in the dorsal horn of the spinal cord. This coincides with behavioral signs of acute inflammatory pain, but whether CP-AMPARs contribute to chronic pain remains unclear. To evaluate this question, we first constructed current-voltage (IV) curves of C-fiber stimulus-evoked, AMPAR-mediated EPSCs in lamina II to test for inward rectification, a key characteristic of CP-AMPARs. We found that the intraplantar injection of complete Freund's adjuvant (CFA) induced an inward rectification at 3 d that persisted to 21 d after injury. Furthermore, the CP- AMPAR antagonist IEM-1460 (50 µM) inhibited AMPAR-evoked Ca2+ transients 21d after injury but had no effect in uninflamed mice. We then used a model of long-lasting vulnerability for chronic pain that is determined by the balance between latent central sensitization (LCS) and mu opioid receptor constitutive activity (MORCA). When administered 21 d after the intraplantar injection of CFA, intrathecal administration of the MORCA inverse agonist naltrexone (NTX, 1 µg, i.t.) reinstated mechanical hypersensitivity, and superfusion of spinal cord slices with NTX (10 µM) increased the peak amplitude of AMPAR-evoked Ca2+ transients in lamina II neurons. The CP-AMPAR antagonist naspm (0-10 nmol, i.t.) inhibited these NTX-induced increases in mechanical hypersensitivity. NTX had no effect in uninflamed mice. Subsequent western blot analysis of the postsynaptic density membrane fraction from lumbar dorsal horn revealed that CFA increased GluA1 expression at 2 d and GluA4 expression at both 2 and 21 d post-injury, indicating that not just the GluA1 subunit, but also the GluA4 subunit, contributes to the expression of CP-AMPARs and synaptic strength during hyperalgesia. GluA2 expression increased at 21 d, an unexpected result that requires further study. We conclude that after tissue injury, dorsal horn AMPARs retain a Ca2+ permeability that underlies LCS. Because of their effectiveness in reducing naltrexone-induced reinstatement of hyperalgesia and potentiation of AMPAR-evoked Ca2+ signals, CP-AMPAR inhibitors are a promising class of agents for the treatment of chronic inflammatory pain.

An NPY Y1 receptor antagonist unmasks latent sensitization and reveals the contribution of protein kinase A and Epac to chronic inflammatory pain.

Peripheral inflammation produces a long-lasting latent sensitization of spinal nociceptive neurons, that is, masked by tonic inhibitory controls. We explored mechanisms of latent sensitization with an established four-step approach: (1) induction of inflammation; (2) allow pain hypersensitivity to resolve; (3) interrogate latent sensitization with a channel blocker, mutant mouse, or receptor antagonist; and (4) disrupt compensatory inhibition with a receptor antagonist so as to reinstate pain hypersensitivity. We found that the neuropeptide Y Y1 receptor antagonist BIBO3304 reinstated pain hypersensitivity, indicative of an unmasking of latent sensitization. BIBO3304-evoked reinstatement was not observed in AC1 knockout mice and was prevented with intrathecal co-administration of a pharmacological blocker to the N-methyl-D-aspartate receptor (NMDAR), adenylyl cyclase type 1 (AC1), protein kinase A (PKA), transient receptor potential cation channel A1 (TRPA1), channel V1 (TRPV1), or exchange protein activated by cAMP (Epac1 or Epac2). A PKA activator evoked both pain reinstatement and touch-evoked pERK expression in dorsal horn; the former was prevented with intrathecal co-administration of a TRPA1 or TRPV1 blocker. An Epac activator also evoked pain reinstatement and pERK expression. We conclude that PKA and Epac are sufficient to maintain long-lasting latent sensitization of dorsal horn neurons that is kept in remission by the NPY-Y1 receptor system. Furthermore, we have identified and characterized 2 novel molecular signaling pathways in the dorsal horn that drive latent sensitization in the setting of chronic inflammatory pain: NMDAR→AC1→PKA→TRPA1/V1 and NMDAR→AC1→Epac1/2. New treatments for chronic inflammatory pain might either increase endogenous NPY analgesia or inhibit AC1, PKA, or Epac.

Fingolimod reduces neuropathic pain behaviors in a mouse model of multiple sclerosis by a sphingosine-1 phosphate receptor 1-dependent inhibition of central sensitization in the dorsal horn.

Multiple sclerosis (MS) is an autoimmune-inflammatory neurodegenerative disease that is often accompanied by a debilitating neuropathic pain. Disease-modifying agents slow down the progression of multiple sclerosis and prevent relapses, yet it remains unclear if they yield analgesia. We explored the analgesic potential of fingolimod (FTY720), an agonist and/or functional antagonist at the sphingosine-1-phosphate receptor 1 (S1PR1), because it reduces hyperalgesia in models of peripheral inflammatory and neuropathic pain. We used a myelin oligodendrocyte glycoprotein 35 to 55 (MOG35-55) mouse model of experimental autoimmune encephalomyelitis, modified to avoid frank paralysis, and thus, allow for assessment of withdrawal behaviors to somatosensory stimuli. Daily intraperitoneal fingolimod reduced behavioral signs of central neuropathic pain (mechanical and cold hypersensitivity) in a dose-dependent and reversible manner. Both autoimmune encephalomyelitis and fingolimod changed hyperalgesia before modifying motor function, suggesting that pain-related effects and clinical neurological deficits were modulated independently. Fingolimod also reduced cellular markers of central sensitization of neurons in the dorsal horn of the spinal cord: glutamate-evoked Ca signaling and stimulus-evoked phospho-extracellular signal-related kinase ERK (pERK) expression, as well as upregulation of astrocytes (GFAP) and macrophage/microglia (Iba1) immunoreactivity. The antihyperalgesic effects of fingolimod were prevented or reversed by the S1PR1 antagonist W146 (1 mg/kg daily, i.p.) and could be mimicked by either repeated or single injection of the S1PR1-selective agonist SEW2871. Fingolimod did not change spinal membrane S1PR1 content, arguing against a functional antagonist mechanism. We conclude that fingolimod behaves as an S1PR1 agonist to reduce pain in multiple sclerosis by reversing central sensitization of spinal nociceptive neurons.

Antinociceptive Effects of the Antidepressant Phenelzine are Mediated by Context-Dependent Inhibition of Neuronal Responses in the Dorsal Horn.

The putative strong anti-nociceptive properties of the antidepressant phenelzine (PLZ) have not been widely explored as a treatment for pain. Antinociceptive effects of PLZ were identified in the formalin model of tonic pain (Mifflin et al., 2016) and in allodynia associated with experimental autoimmune encephalomyelitis, (EAE) a mouse model of multiple sclerosis (Potter et al., 2016). Here, we further clarify the specific types of stimuli and contexts in which PLZ modulates nociceptive sensitivity. Our findings indicate that PLZ selectively inhibits ongoing inflammatory pain while sparing transient reflexive and acute nociception. We also investigated the cellular mechanisms of action of PLZ in the dorsal horn, and as expected of a monoamine-oxidase inhibitor, PLZ increased serotonin (5HT) immunoreactivity. We next used two approaches to test the hypothesis that PLZ inhibits the activation of spinal nociresponsive neurons. First, we evaluated the formalin-evoked protein expression of the immediate early gene, c-fos. PLZ reduced Fos expression in the superficial dorsal horn. Second, we evaluated the effects of PLZ on intracellular calcium responses to superfusion of glutamate (0.3-1.0 mM) in an ex vivo lumbar spinal cord slice preparation. Superfusion with PLZ (100-300 µM) reduced 1 mM glutamate-evoked calcium responses. This was blocked by pretreatment with the 5HT1A-receptor antagonist WAY-100,635, but not the alpha-2 adrenergic antagonist idazoxan. We conclude that PLZ exerts antinociceptive effects through a 5-HT/5HT1AR-dependent inhibition of neuronal responses within nociceptive circuits of the dorsal horn.

Photochemical scar ablation in chronically contused spinal cord of rat.

Glial scar represents a physical and molecular barrier to axonal regeneration and has become an important target for regeneration research in chronic spinal cord injury. Although many methods have been proven useful for the prevention of scar formation in an acute injury model, to date no effective method has been described to remove an existing glial scar in a chronic injury. The chronic lesion possesses an irregular shaped scar that lines the entire perimeter of the cavity. In the present study, we used rose bengal, a molecule commonly used for biological staining, injected into the cavity at the injury site of Long-Evans rat spinal cord (5 weeks after 25-mm contusion injury). Visible light was used to illuminate the injury site. Histological observation illustrates that at least partial glial scar tissue is ablated by rose bengal/illumination. The lack of glial fibrillary acidic protein (GFAP) immunoreactivity at the glial scar coupled with the reduction of GFAP density surrounding spared tissue suggests that this photochemical scar ablation preferentially kills astrocytes at the scar tissue but also reacts, to a lesser degree, in the spared tissue. There is an observed reduction of Basso, Beattie, and Bresnahan (BBB) scale scores after scar ablation, but it is not statistically significant from stabilized behavioral scoring prior to the scar ablation treatment. Our findings indicate that the rose bengal/illumination is feasible for ablation of the glial scar which surrounds an irregular lesion cavity in shape. The scar ablation might provide a permissive environment for the regenerating axons when enriched by cellular or drug therapy.

Simvastatin promotes neurite outgrowth in the presence of inhibitory molecules found in central nervous system injury.

Statins (3-hydroxy-3-methylglutaryl-CoA [HMG-CoA] reductase inhibitors) inhibit the rate-limiting step in the mevalonate pathway, conversion of HMG-CoA to mevalonate, by competitive inhibition with the enzyme HMG-CoA reductase. Statins not only lower cholesterol levels, but are also thought to exert neuroprotective and neurogenic effects that may be beneficial in treating brain and spinal cord injuries. Data presented here illustrate that simvastatin enables neurite outgrowth in the presence of growth-inhibitory molecules commonly found at central nervous system (CNS) injury sites. To assess the effect of simvastatin on neurite outgrowth in the presence of inhibitory molecules present at CNS injury sites, rat embryonic cortex explants or postnatal spinal cord explants were grown on membrane filters prepared with alternating stripes of laminin and myelin/laminin. Immunostaining indicated that myelin stripes contain myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and Nogo, but do not contain chondroitin sulfate proteoglycan (CSPG). When control explants were grown in the presence of alternating stripes, neurite outgrowth preferentially extended in regions containing laminin only. In contrast, neurite outgrowth from explants grown in the presence of simvastatin was significantly less selective for laminin regions and was able to extend into regions containing myelin (p < 0.01). Simvastatin-induced effects were reversed by addition of mevalonate. Isoprenyl transferase inhibitors GGTI-286 and FTI-277, inhibitors of biochemical steps subsequent to HMG-CoA conversion to mevalonate, mimicked simvastatin- induced effects. These data suggest that simvastatin counteracts myelin-associated neurite outgrowth inhibition signals via mevalonate pathway inhibition, and may be beneficial in promoting axon regeneration in brain and spinal cord injury.

Conventional protein kinase C isoforms regulate human dopamine transporter activity in Xenopus oocytes.

The hypothesis that specific protein kinase C (PKC) isoforms regulate dopamine transporter (DAT) function was tested in Xenopus laevis oocytes expressing human (h)DAT. Activation of conventional PKCs (cPKCs) and novel PKCs (nPKCs) using 10 nM phorbol 12-myristate 13-acetate (PMA) significantly inhibited DAT-associated transport currents. This effect was reversed by isoform-non-selective PKC inhibitors, selective inhibitors of cPKCs and deltaPKC, and by Ca2+ chelation. By contrast, the epsilonPKC translocation inhibitor peptide had no effect on PMA-induced inhibition of hDAT transport-associated currents. Thus, the primary mechanism by which PMA regulates hDAT expressed in oocytes appears to be by activating cPKC(s).

Statins decrease chondroitin sulfate proteoglycan expression and acute astrocyte activation in central nervous system injury.

Statins elicit numerous favorable effects on central nervous system (CNS) injury, including inhibition of the rhoA/ROCK pathway. In the present study, we show that statins decrease acute astrocyte activation in CNS injury, and decrease chondroitin sulfate proteoglycan (CSPG) levels in astrocyte cultures as well as CNS injury. CSPG levels decreased by up to 45% in simvastatin-treated astrocyte cultures compared to control cultures. In simvastatin-treated animals, CSPG levels declined by 60% 8 days after brain stab injury, and by 62-64% 4 weeks after spinal cord injury (SCI). Glial fibrillary acid protein (GFAP) levels decreased in brain stab at 8 days after surgery/intervention, suggesting that statins produce a decrease in astrocyte activation. Attenuation of astrocyte activation may contribute to the decline in CSPG levels. However, there are likely other contributing factors, since GFAP levels were not a contributing factor in the decline of CSPG levels in astrocyte cultures. Robust locomotor improvements were not observed with any treatment. The numerous beneficial effects of statins on CNS injury render them an attractive candidate in the treatment of CNS injury.

Brief, repeated exposure to substrates down-regulates dopamine transporter function in Xenopus oocytes in vitro and rat dorsal striatum in vivo.

In heterologous expression systems, dopamine transporter (DAT) cell-surface localization is reduced after relatively prolonged exposure to d-amphetamine (AMPH) or dopamine (DA), suggesting a role for substrate-mediated regulation of transporter function. Here, we investigated whether brief, repeated periods of substrate exposure modulated transporter function, first, in an in vitro model system and, second, in intact rat brain. In human DAT-expressing Xenopus laevis oocytes, repeated exposure to low micromolar concentrations of DA, AMPH or tyramine markedly reduced transport-mediated currents. This functional down-regulation was attenuated by inclusion of a protein kinase C (PKC) inhibitor and probably reflects DAT redistribution, as cell-surface [3H]WIN 35 428 binding was significantly lower following DA exposure. High-speed chronoamperometry was used to measure clearance of exogenously applied DA in dorsal striatum (STR) and nucleus accumbens (NAc) of anesthetized rats. In STR, frequent (every 2 min) applications of DA altered DA clearance parameters in a manner consistent with profound down-regulation of DAT function. Similar changes were not observed in NAc or after repeated vehicle (ascorbic acid) application. Together, our results suggest that brief, repeated periods of substrate exposure lead to rapid down-regulation of DAT activity and that this type of regulation can occur in vivo in STR, but not NAc.

Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy.

To examine the oligomeric state and trafficking of the dopamine transporter (DAT) in different compartments of living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP). YFP-DAT and CFP-DAT were transiently and stably expressed in porcine aortic endothelial (PAE) cells, human embryonic kidney (HEK) 293 cells, and an immortalized dopaminergic cell line 1RB3AN27. Fluorescence microscopic imaging of cells co-expressing YFP-DAT and CFP-DAT revealed fluorescence resonance energy transfer (FRET) between CFP and YFP, which is consistent with an intermolecular interaction of DAT fusion proteins. FRET signals were detected between CFP- and YFP-DAT located at the plasma membrane and in intracellular membrane compartments. Phorbol esters or amphetamine induced the endocytosis of YFP/CFP-DAT to early and recycling endosomes, identified by Rab5, Rab11, Hrs and EEA.1 proteins. Interestingly, however, DAT was mainly excluded from Rab5- and Hrs-containing microdomains within the endosomes. The strongest FRET signals were measured in endosomes, indicative of efficient oligomerization of internalized DAT. The intermolecular DAT interactions were confirmed by co-immunoprecipitation. A DAT mutant that was retained in the endoplasmic reticulum (ER) after biosynthesis was used to show that DAT is oligomeric in the ER. Moreover, co-expression of an ER-retained DAT mutant and wild-type DAT resulted in the retention of wild-type DAT in the ER. These data suggest that DAT oligomers are formed in the ER and then are constitutively maintained both at the cell surface and during trafficking between the plasma membrane and endosomes.

MT(2) melatonin receptors are present and functional in rat caudal artery.

In rat caudal artery, contraction to melatonin results primarily from activation of MT(1) melatonin receptors; however, the role of MT(2) melatonin receptors in vascular responses is controversial. We examined and compared the expression and function of MT(2) receptors with that of MT(1) receptors in male rat caudal artery. MT(1) and MT(2) melatonin receptor mRNA was amplified by reverse transcription-polymerase chain reaction from caudal arteries of three rat strains (i.e., Fisher, Sprague-Dawley, and Wistar). Antisense (but not sense) (33)P-labeled oligonucleotide probes specific for MT(1) or MT(2) receptor mRNA hybridized to smooth muscle, as well as intimal and adventitial layers, of caudal artery. In male Fisher rat caudal artery denuded of endothelium, melatonin was 10 times more potent than 6-chloromelatonin to potentiate contraction to phenylephrine, suggesting activation of smooth muscle MT(1) melatonin receptors. The MT(1)/MT(2) competitive melatonin receptor antagonist luzindole (3 microM), blocked melatonin-mediated contraction (0.1-100 nM) with an affinity constant (K(B) value of 157 nM) similar to that for the human MT(1) receptor. However, at melatonin concentrations above 100 nM, luzindole potentiated the contractile response, suggesting blockade of MT(2) receptors mediating vasorelaxation and/or an inverse agonist effect at MT(1) constitutively active receptors. The involvement of MT(2) receptors in vasorelaxation is supported by the finding that the competitive antagonists 4-phenyl 2-acetamidotetraline and 4-phenyl-2-propionamidotetraline, at MT(2)-selective concentrations (10 nM), significantly enhanced contractile responses to all melatonin concentrations tested (0.1 nM-10 microM). We conclude that MT(2) melatonin receptors expressed in vascular smooth muscle mediate vasodilation in contrast to vascular MT(1) receptors mediating vasoconstriction.