Four sulfur-containing compounds with anti-colon cancer effect from marine-derived fungus Aspergillus terreus.

Sulfur-containing natural products possess a variety of biological functions including antitumor, antibacterial, anti-inflammatory and antiviral activities. In this study, four previously undescribed sulfur-containing compounds asperteretals L and M, terreins A and B, together with 17 known compounds were obtained from a culture of marine fungus A. terreus supplemented with inorganic sulfur source Na2SO4. Their planar structures and absolute configurations were elucidated by NMR, HRESIMS, and ECD experiments. The in vitro cytotoxicities of compounds 1-21 against HCT-116 and Caco-2 were evaluated by SRB assay. Asperteretal M (2) exhibited activity against HCT-116 with the IC50 value at 30µM. The antiproliferative effect of asperteretal M was confirmed by colony formation assay and cell death staining. Furthermore, the preliminary study on the anti-colon cancer mechanism of asperteretal M was performed by RNA-seq analysis. Western blotting validated that asperteretal M significantly decreased the expression of cell-cycle regulatory proteins CDK1, CDK4, and PCNA in a concentration-dependent manner.

mGluR5 from Primary Sensory Neurons Promotes Opioid-Induced Hyperalgesia and Tolerance by Interacting with and Potentiating Synaptic NMDA Receptors.

Aberrant activation of presynaptic NMDARs in the spinal dorsal horn is integral to opioid-induced hyperalgesia and analgesic tolerance. However, the signaling mechanisms responsible for opioid-induced NMDAR hyperactivity remain poorly identified. Here, we show that repeated treatment with morphine or fentanyl reduced monomeric mGluR5 protein levels in the dorsal root ganglion (DRG) but increased levels of mGluR5 monomers and homodimers in the spinal cord in mice and rats of both sexes. Coimmunoprecipitation analysis revealed that monomeric and dimeric mGluR5 in the spinal cord, but not monomeric mGluR5 in the DRG, directly interacted with GluN1. By contrast, mGluR5 did not interact with µ-opioid receptors in the DRG or spinal cord. Repeated morphine treatment markedly increased the mGluR5-GluN1 interaction and protein levels of mGluR5 and GluN1 in spinal synaptosomes. The mGluR5 antagonist MPEP reversed morphine treatment-augmented mGluR5-GluN1 interactions, GluN1 synaptic expression, and dorsal root-evoked monosynaptic EPSCs of dorsal horn neurons. Furthermore, CRISPR-Cas9-induced conditional mGluR5 knockdown in DRG neurons normalized mGluR5 levels in spinal synaptosomes and NMDAR-mediated EPSCs of dorsal horn neurons increased by morphine treatment. Correspondingly, intrathecal injection of MPEP or conditional mGluR5 knockdown in DRG neurons not only potentiated the acute analgesic effect of morphine but also attenuated morphine treatment-induced hyperalgesia and tolerance. Together, our findings suggest that opioid treatment promotes mGluR5 trafficking from primary sensory neurons to the spinal dorsal horn. Through dimerization and direct interaction with NMDARs, presynaptic mGluR5 potentiates and/or stabilizes NMDAR synaptic expression and activity at primary afferent central terminals, thereby maintaining opioid-induced hyperalgesia and tolerance.SIGNIFICANCE STATEMENT Opioids are essential analgesics for managing severe pain caused by cancer, surgery, and tissue injury. However, these drugs paradoxically induce pain hypersensitivity and tolerance, which can cause rapid dose escalation and even overdose mortality. This study demonstrates, for the first time, that opioids promote trafficking of mGluR5, a G protein-coupled glutamate receptor, from peripheral sensory neurons to the spinal cord; there, mGluR5 proteins dimerize and physically interact with NMDARs to augment their synaptic expression and activity. Through dynamic interactions, the two distinct glutamate receptors mutually amplify and sustain nociceptive input from peripheral sensory neurons to the spinal cord. Thus, inhibiting mGluR5 activity or disrupting mGluR5-NMDAR interactions could reduce opioid-induced hyperalgesia and tolerance and potentiate opioid analgesic efficacy.

NMDA Receptors at Primary Afferent-Excitatory Neuron Synapses Differentially Sustain Chemotherapy- and Nerve Trauma-Induced Chronic Pain.

The spinal dorsal horn contains vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons and vesicular GABA transporter (VGAT)-expressing inhibitory neurons, which normally have different roles in nociceptive transmission. Spinal glutamate NMDAR hyperactivity is a crucial mechanism of chronic neuropathic pain. However, it is unclear how NMDARs regulate primary afferent input to spinal excitatory and inhibitory neurons in neuropathic pain. Also, the functional significance of presynaptic NMDARs in neuropathic pain has not been defined explicitly. Here we showed that paclitaxel treatment or spared nerve injury (SNI) similarly increased the NMDAR-mediated mEPSC frequency and dorsal root-evoked EPSCs in VGluT2 dorsal horn neurons in male and female mice. By contrast, neither paclitaxel nor SNI had any effect on mEPSCs or evoked EPSCs in VGAT neurons. In mice with conditional Grin1 (gene encoding GluN1) KO in primary sensory neurons (Grin1-cKO), paclitaxel treatment failed to induce pain hypersensitivity. Unexpectedly, SNI still caused long-lasting pain hypersensitivity in Grin1-cKO mice. SNI increased the amplitude of puff NMDA currents in VGluT2 neurons and caused similar depolarizing shifts in GABA reversal potentials in WT and Grin1-cKO mice. Concordantly, spinal Grin1 knockdown diminished SNI-induced pain hypersensitivity. Thus, presynaptic NMDARs preferentially amplify primary afferent input to spinal excitatory neurons in neuropathic pain. Although presynaptic NMDARs are required for chemotherapy-induced pain hypersensitivity, postsynaptic NMDARs in spinal excitatory neurons play a dominant role in traumatic nerve injury-induced chronic pain. Our findings reveal the divergent synaptic connectivity and functional significance of spinal presynaptic and postsynaptic NMDARs in regulating cell type-specific nociceptive input in neuropathic pain with different etiologies.SIGNIFICANCE STATEMENT Spinal excitatory neurons relay input from nociceptors, whereas inhibitory neurons repress spinal nociceptive transmission. Chronic nerve pain is associated with aberrant NMDAR activity in the spinal dorsal horn. This study demonstrates, for the first time, that chemotherapy and traumatic nerve injury preferentially enhance the NMDAR activity at primary afferent-excitatory neuron synapses but have no effect on primary afferent input to spinal inhibitory neurons. NMDARs in primary sensory neurons are essential for chemotherapy-induced chronic pain, whereas nerve trauma causes pain hypersensitivity predominantly via postsynaptic NMDARs in spinal excitatory neurons. Thus, presynaptic and postsynaptic NMDARs at primary afferent-excitatory neuron synapses are differentially engaged in chemotherapy- and nerve injury-induced chronic pain and could be targeted respectively for treating these painful conditions.

Indole alkaloids fusarindoles A-E from marine-derived fungus Fusarium equiseti LJ-1.

Five undescribed indole alkaloids, fusarindoles A-E, together with seven known compounds were obtained from the marine-derived fungus Fusarium equiseti LJ-1. Their chemical structures and absolute configurations were determined by comprehensive analysis of the NMR, HRMS, UV, IR, ECD calculation and single-crystal X-ray diffraction data. The possible biosynthetic pathways of fusarindoles C-E were proposed. The cytotoxicities of eleven compounds, including fusarindoles A-E and six known compounds, against five human cancer cell lines A549, CNE2, SUNE1, HepG2 and QGY7701 were evaluated.

α2δ-1 protein promotes synaptic expression of Ca2+ permeable-AMPA receptors by inhibiting GluA1/GluA2 heteromeric assembly in the hypothalamus in hypertension.

Glutamate AMPA receptors (AMPARs) lacking GluA2 subunit are calcium permeable (CP-AMPARs), which are increased in the hypothalamic paraventricular nucleus (PVN) and maintain sympathetic outflow in hypertension. Here, we determined the role of α2δ-1, an NMDA receptor-interacting protein, in regulating synaptic CP-AMPARs in the hypothalamus in spontaneously hypertensive rats (SHR). Co-immunoprecipitation showed that levels of GluA1/GluA2, but not GluA2/GluA3, protein complexes in hypothalamic synaptosomes were reduced in SHR compared with Wistar-Kyoto rats (WKY). The level of GluA1/GluA2 heteromers in endoplasmic reticulum-enriched fractions of the hypothalamus was significantly lower in SHR than in WKY, which was restored by inhibiting α2δ-1 with gabapentin. Gabapentin also switched AMPAR-mediated excitatory postsynaptic currents (AMPAR-EPSCs) from inward rectifying to linear and attenuated the inhibitory effect of IEM-1460, a selective CP-AMPAR blocker, on AMPAR-EPSCs in spinally projecting PVN neurons in SHR. Furthermore, co-immunoprecipitation revealed that α2δ-1 directly interacted with GluA1 and GluA2 in the hypothalamus of rats and humans. Levels of α2δ-1/GluA1 and α2δ-1/GluA2 protein complexes in the hypothalamus were significantly greater in SHR than in WKY. Disrupting the α2δ-1-AMPAR interaction with an α2δ-1 C terminus peptide normalized GluA1/GluA2 heteromers in the endoplasmic reticulum of the hypothalamus diminished in SHR. In addition, α2δ-1 C terminus peptide diminished inward rectification of AMPAR-EPSCs and the inhibitory effect of IEM-1460 on AMPAR-EPSCs of PVN neurons in SHR. Thus, α2δ-1 augments synaptic CP-AMPARs by inhibiting GluA1/GluA2 heteromeric assembly in the hypothalamus in hypertension. These findings extend our understanding of the molecular basis of sustained sympathetic outflow in neurogenic hypertension.

α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly.

Many neurological disorders show an increased prevalence of GluA2-lacking, Ca2+-permeable AMPA receptors (CP-AMPARs), which dramatically alters synaptic function. However, the molecular mechanism underlying this distinct synaptic plasticity remains enigmatic. Here, we show that nerve injury potentiates postsynaptic, but not presynaptic, CP-AMPARs in the spinal dorsal horn via α2δ-1. Overexpressing α2δ-1, previously regarded as a Ca2+ channel subunit, augments CP-AMPAR levels at the cell surface and synapse. Mechanistically, α2δ-1 physically interacts with both GluA1 and GluA2 via its C terminus, inhibits the GluA1/GluA2 heteromeric assembly, and increases GluA2 retention in the endoplasmic reticulum. Consequently, α2δ-1 diminishes the availability and synaptic expression of GluA1/GluA2 heterotetramers in the spinal cord in neuropathic pain. Inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-AMPAR complex fully restores the intracellular assembly and synaptic dominance of heteromeric GluA1/GluA2 receptors. Thus, α2δ-1 is a pivotal AMPAR-interacting protein that controls the subunit composition and Ca2+ permeability of postsynaptic AMPARs.

Streptozotocin-Induced Diabetic Neuropathic Pain Is Associated with Potentiated Calcium-Permeable AMPA Receptor Activity in the Spinal Cord.

Neuronal hyperactivity in the spinal dorsal horn can amplify nociceptive input in diabetic neuropathic pain. The glutamate N-methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (NMDA receptors and AMPA receptors, respectively) are involved in spinal nociceptive transmission. It is unclear, however, whether painful diabetic neuropathy is associated with changes in the activity of synaptic NMDA receptors and AMPA receptors in spinal dorsal horn neurons. AMPA receptors lacking GluA2 are Ca2+-permeable (CP-AMPA receptors), and their currents display characteristic inward rectification. In this study, we showed that evoked excitatory postsynaptic currents (EPSCs), induced by streptozotocin, exhibited inward rectification in spinal dorsal neurons in diabetic rats. Presynaptic and postsynaptic NMDA receptor activity in the spinal dorsal horn was similar in diabetic and control rats. In the dorsal spinal cord, the membrane GluA2 protein level was significantly lower in diabetic than in control rats, whereas the cytosolic GluA2 level was greater in diabetic than in control rats. In contrast, the GluA1 subunit levels in the plasma membrane and cytosol did not differ between the two groups. Blocking CP-AMPA receptors significantly reduced the amplitude of EPSCs of dorsal horn neurons in diabetic but not in control rats. Furthermore, blocking spinal CP-AMPA receptors reduced pain hypersensitivity in diabetic rats but had no effect on nociception in control rats. Our study suggests that diabetic neuropathy augments CP-AMPA receptor activity in the spinal dorsal horn by causing intracellular retention of GluA2 and impairing GluA2 membrane trafficking. Increased prevalence of spinal CP-AMPA receptors sustains diabetic neuropathic pain. SIGNIFICANCE STATEMENT: This study demonstrates that the prevalence of synaptic calcium-permeable AMPA receptors is increased in the spinal dorsal horn, which mediates pain hypersensitivity in diabetic neuropathy. Thus, calcium-permeable AMPA receptors play an important role in glutamatergic synaptic plasticity in the spinal cord in painful diabetic neuropathy. This new knowledge improves our understanding of the mechanisms involved in central sensitization associated with diabetic neuropathic pain and suggests that calcium-permeable AMPA receptors are an alternative therapeutic target for treating this chronic pain condition.

AMPK activation attenuates inflammatory pain through inhibiting NF-κB activation and IL-1ß expression.

BACKGROUND: Chronic pain is a major clinical problem with limited treatment options. Previous studies have demonstrated that activation of adenosine monophosphate-activated protein kinase (AMPK) can attenuate neuropathic pain. Inflammation/immune response at the site of complete Freund's adjuvant (CFA) injection is known to be a critical trigger of the pathological changes that produce inflammatory pain. However, whether activation of AMPK produces an analgesic effect through inhibiting the proinflammatory cytokines, including interleukin-1ß (IL-1ß), in inflammatory pain remains unknown. METHODS: Inflammatory pain was induced in mice injected with CFA. The effects of AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside, an AMPK activator), Compound C (an AMPK inhibitor), and IL-1ra (an IL-1 receptor antagonist) were tested at day 4 after CFA injection. Inflammatory pain was assessed with von Frey filaments and hot plate. Immunoblotting, hematoxylin and eosin (H&E) staining, and immunofluorescence were used to assess inflammation-induced biochemical changes. RESULTS: The AMPK activator AICAR produced an analgesic effect and inhibited the level of proinflammatory cytokine IL-1ß in the inflamed skin in mice. Moreover, activation of AMPK suppressed CFA-induced NF-κB p65 translocation from the cytosol to the nucleus in activated macrophages (CD68+ and CX3CR1+) of inflamed skin tissues. Subcutaneous injection of IL-1ra attenuated CFA-induced inflammatory pain. The AMPK inhibitor Compound C and AMPKα shRNA reversed the analgesic effect of AICAR and the effects of AICAR on IL-1ß and NF-κB activation in inflamed skin tissues. CONCLUSIONS: Our study provides new information that AMPK activation produces the analgesic effect by inhibiting NF-κB activation and reducing the expression of IL-1ß in inflammatory pain.

Increased α2δ-1-NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain.

Painful peripheral neuropathy is a severe and difficult-to-treat neurological complication associated with cancer chemotherapy. Although chemotherapeutic drugs such as paclitaxel are known to cause tonic activation of presynaptic NMDA receptors (NMDARs) to potentiate nociceptive input, the molecular mechanism involved in this effect is unclear. α2δ-1, commonly known as a voltage-activated calcium channel subunit, is a newly discovered NMDAR-interacting protein and plays a critical role in NMDAR-mediated synaptic plasticity. Here we show that paclitaxel treatment in rats increases the α2δ-1 expression level in the dorsal root ganglion and spinal cord and the mRNA levels of GluN1, GluN2A, and GluN2B in the spinal cord. Paclitaxel treatment also potentiates the α2δ-1-NMDAR interaction and synaptic trafficking in the spinal cord. Strikingly, inhibiting α2δ-1 trafficking with pregabalin, disrupting the α2δ-1-NMDAR interaction with an α2δ-1 C-terminus-interfering peptide, or α2δ-1 genetic ablation fully reverses paclitaxel treatment-induced presynaptic NMDAR-mediated glutamate release from primary afferent terminals to spinal dorsal horn neurons. In addition, intrathecal injection of pregabalin or α2δ-1 C-terminus-interfering peptide and α2δ-1 knockout in mice markedly attenuate paclitaxel-induced pain hypersensitivity. Our findings indicate that α2δ-1 is required for paclitaxel-induced tonic activation of presynaptic NMDARs at the spinal cord level. Targeting α2δ-1-bound NMDARs, not the physiological α2δ-1-free NMDARs, may be a new strategy for treating chemotherapy-induced neuropathic pain. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Regulating nociceptive transmission by VGluT2-expressing spinal dorsal horn neurons.

Vesicular glutamate transporter-2 (VGluT2) mediates the uptake of glutamate into synaptic vesicles in neurons. Spinal cord dorsal horn interneurons are highly heterogeneous and molecularly diverse. The functional significance of VGluT2-expressing dorsal horn neurons in physiological and pathological pain conditions has not been explicitly demonstrated. Designer receptors exclusively activated by designer drugs (DREADDs) are a powerful chemogenetic tool to reversibly control neuronal excitability and behavior. Here, we used transgenic mice with Cre recombinase expression driven by the VGluT2 promoter, combined with the chemogenetic approach, to determine the contribution of VGluT2-expressing dorsal horn neurons to nociceptive regulation. Adeno-associated viral vectors expressing double-floxed Cre-dependent Gαq-coupled human M3 muscarinic receptor DREADD (hM3D)-mCherry or Gαi-coupled κ-opioid receptor DREADD (KORD)-IRES-mCitrine were microinjected into the superficial spinal dorsal horn of VGluT2-Cre mice. Immunofluorescence labeling showed that VGluT2 was predominantly expressed in lamina II excitatory interneurons. Activation of excitatory hM3D in VGluT2-expressing neurons with clozapine N-oxide caused a profound increase in neuronal firing and synaptic glutamate release. Conversely, activation of inhibitory KORD in VGluT2-expressing neurons with salvinorin B markedly inhibited neuronal activity and synaptic glutamate release. In addition, chemogenetic stimulation of VGluT2-expressing neurons increased mechanical and thermal sensitivities in naive mice, whereas chemogenetic silencing of VGluT2-expressing neurons reversed pain hypersensitivity induced by tissue inflammation and peripheral nerve injury. These findings indicate that VGluT2-expressing excitatory neurons play a crucial role in mediating nociceptive transmission in the spinal dorsal horn. Targeting glutamatergic dorsal horn neurons with inhibitory DREADDs may be a new strategy for treating inflammatory pain and neuropathic pain.

Presynaptic mGluR5 receptor controls glutamatergic input through protein kinase C-NMDA receptors in paclitaxel-induced neuropathic pain.

Chemotherapeutic drugs such as paclitaxel cause painful peripheral neuropathy in many cancer patients and survivors. Although NMDA receptors (NMDARs) at primary afferent terminals are known to be critically involved in chemotherapy-induced chronic pain, the upstream signaling mechanism that leads to presynaptic NMDAR activation is unclear. Group I metabotropic glutamate receptors (mGluRs) play a role in synaptic plasticity and NMDAR regulation. Here we report that the Group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) significantly increased the frequency of miniature excitatory postsynaptic currents (EPSCs) and the amplitude of monosynaptic EPSCs evoked from the dorsal root. DHPG also reduced the paired-pulse ratio of evoked EPSCs in spinal dorsal horn neurons. These effects were blocked by the selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP), but not by an mGluR1 antagonist. MPEP normalized the frequency of miniature EPSCs and the amplitude of evoked EPSCs in paclitaxel-treated rats but had no effect in vehicle-treated rats. Furthermore, mGluR5 protein levels in the dorsal root ganglion and spinal cord synaptosomes were significantly higher in paclitaxel- than in vehicle-treated rats. Inhibiting protein kinase C (PKC) or blocking NMDARs abolished DHPG-induced increases in the miniature EPSC frequency of spinal dorsal horn neurons in vehicle- and paclitaxel-treated rats. Moreover, intrathecal administration of MPEP reversed pain hypersensitivity caused by paclitaxel treatment. Our findings suggest that paclitaxel-induced painful neuropathy is associated with increased presynaptic mGluR5 activity at the spinal cord level, which serves as upstream signaling for PKC-mediated tonic activation of NMDARs. mGluR5 is therefore a promising target for reducing chemotherapy-induced neuropathic pain.

Bortezomib induces neuropathic pain through protein kinase C-mediated activation of presynaptic NMDA receptors in the spinal cord.

Chemotherapeutic drugs, including bortezomib, often cause painful peripheral neuropathy, which is a severe dose-limiting adverse effect experienced by many cancer patients. The glutamate N-methyl-d-aspartate receptors (NMDARs) at the spinal cord level are critically involved in the synaptic plasticity associated with neuropathic pain. In this study, we determined whether treatment with bortezomib, a proteasome inhibitor, affects the NMDAR activity of spinal dorsal horn neurons. Systemic treatment with bortezomib in rats did not significantly affect postsynaptic NMDAR currents elicited by puff application of NMDA directly to dorsal horn neurons. Bortezomib treatment markedly increased the baseline frequency of miniature excitatory postsynaptic currents (EPSCs), which was completely normalized by the NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). AP5 also reduced the amplitude of monosynaptic EPSCs evoked by dorsal root stimulation in bortezomib-treated, but not vehicle-treated, rats. Furthermore, inhibition of protein kinase C (PKC) with chelerythrine fully reversed the increased frequency of miniature EPSCs and the amplitude of evoked EPSCs in bortezomib-treated rats. Intrathecal injection of AP5 and chelerythrine both profoundly attenuated mechanical allodynia and hyperalgesia induced by systemic treatment with bortezomib. In addition, treatment with bortezomib induced striking membrane translocation of PKC-ßII, PKC-δ, and PKC-ε in the dorsal root ganglion. Our findings indicate that bortezomib treatment potentiates nociceptive input from primary afferent nerves via PKC-mediated tonic activation of presynaptic NMDARs. Targeting presynaptic NMDARs and PKC at the spinal cord level may be an effective strategy for treating chemotherapy-induced neuropathic pain.

Peripheral Motor and Sensory Nerve Conduction following Transplantation of Undifferentiated Autologous Adipose Tissue-Derived Stem Cells in a Biodegradable U.S. Food and Drug Administration-Approved Nerve Conduit.

BACKGROUND: Conduits preseeded with either Schwann cells or stem cells differentiated into Schwann cells demonstrated promising results for the outcome of nerve regeneration in nerve defects. METHODS: The concept of this trial combines nerve repair by means of a commercially available nerve guidance conduit and preseeding with autologous, undifferentiated, adipose tissue-derived stem cells. Adipose tissue-derived stem cells were harvested from rats and subsequently seeded onto a U.S. Food and Drug Administration-approved type I collagen conduit. Sciatic nerve gaps 10 mm in length were created, and nerve repair was performed by the transplantation of either conduits preseeded with autologous adipose tissue-derived stem cells or acellular (control group) conduits. After 6 months, the motor and sensory nerve conduction velocity were assessed. Nerves were removed and examined by hematoxylin and eosin, van Gieson, and immunohistochemistry (S100 protein) staining for the quality of axonal regeneration. RESULTS: Nerve gaps treated with adipose tissue-derived stem cells showed superior nerve regeneration, reflected by higher motor and sensory nerve conduction velocity values. The motor and sensory nerve conduction velocity were significantly greater in nerves treated with conduits preseeded with adipose tissue-derived stem cells than in nerves treated with conduits alone (p < 0.05). Increased S100 immunoreactivity was detected for the adipose tissue-derived stem cell group. In this group, axon arrangement inside the conduits was more organized. CONCLUSIONS: Transplantation of adipose tissue-derived stem cells significantly improves motor and sensory nerve conduction velocity in peripheral nerve gaps. Preseeded conduits showed a more organized axon arrangement inside the conduit in comparison with nerve conduits alone. The approach used here could readily be translated into a clinical therapy. CLINCAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Netrin-1 Contributes to Myelinated Afferent Fiber Sprouting and Neuropathic Pain.

Netrin-1 is a neuronal guidance molecule implicated in the development of spinal cord neurons and cortical neurons. In the adult spinal cord, UNC5H (repulsive receptor of netrin-1), but not deleted in colorectal cancer (DCC) (attractive receptor of netrin-1), constitutes a major mode of netrin-1 signal transduction, which may be involved in axon repulsion and inhibits neurite outgrowth. Abnormal sprouting of myelinated afferent fibers in the spinal dorsal horn can cause mechanical allodynia associated with postherpetic neuralgia (PHN, Shingles) and other neuropathic pains. However, whether netrin-1 participates in sprouting of myelinated afferent fibers and mechanical allodynia remains unknown. In an ultropotent TRPV1 agonist resiniferatoxin (RTX)-induced PHN-like model, RTX treatment for 6 weeks increased netrin-1 expression in dorsal horn neurons, including NK-1-positive projection neurons. In human neuroblastoma SH-SY5Y cells, we found that TRPV1 antagonist capsazepine antagonized RTX-induced upregulation of netrin-1. After RTX treatment, UNC5H2 expression was gradually decreased, whereas DCC expression was significantly increased. Silencing netrin-1 in the spinal dorsal horn significantly attenuated RTX-induced mechanical allodynia and sprouting of myelinated fibers into the spinal lamina II. Our results suggest that RTX treatment upregulates netrin-1 expression through activation of TRPV1 receptors and change UNC5H2-rich spinal dorsal horn into a growth-permissive environment by increasing DCC expression, thus enhancing the sprouting of myelinated afferent nerves. Netrin-1 may be targeted for reducing primary afferent sprouting and mechanical allodynia in PHN and other neuropathic pain conditions.

Evaluating the use of antibiotic prophylaxis during open reduction and internal fixation surgery in patients at low risk of surgical site infection.

BACKGROUND: Widespread overuse and inappropriate use of antibiotics contribute to increasingly antibiotic-resistant pathogens and higher health care costs. It is not clear whether routine antibiotic prophylaxis can reduce the rate of surgical site infection (SSI) in low-risk patients undergoing orthopaedic surgery. We designed a simple scorecard to grade SSI risk factors and determined whether routine antibiotic prophylaxis affects SSI occurrence during open reduction and internal fixation (ORIF) orthopaedic surgeries in trauma patients at low risk of developing SSI. METHODS: The SSI risk scorecard (possible total points ranged from 5 to 25) was designed to take into account a patient's general health status, the primary cause of fractures, surgical site tissue condition or wound class, types of devices implanted, and surgical duration. Patients with a low SSI risk score (≤8 points) who were undergoing clean ORIF surgery were divided into control (routine antibiotic treatment, cefuroxime) and evaluation (no antibiotic treatment) groups and followed up for 13-17 months after surgery. RESULTS: The infection rate was much higher in patients with high SSI risk scores (≥9 points) than in patients with low risk scores assigned to the control group (10.7% vs. 2.2%, P<0.0001). SSI occurred in 11 of 499 patients in the control group and in 13 of 534 patients in the evaluation group during the follow-up period of 13-17 months. The SSI occurrence rate did not differ significantly (2.2% vs. 2.4%, P=0.97) between the control and evaluation groups. CONCLUSIONS: Routine antibiotic prophylaxis does not significantly decrease the rate of SSI in ORIF surgical patients with a low risk score. Implementation of this scoring system could guide the rational use of perioperative antibiotics and ultimately reduce antibiotic resistance, health care costs, and adverse reactions to antibiotics.

Hyper-SUMOylation of the Kv7 potassium channel diminishes the M-current leading to seizures and sudden death.

Sudden unexplained death in epilepsy (SUDEP) is the most common cause of premature mortality in epilepsy and was linked to mutations in ion channels; however, genes within the channel protein interactome might also represent pathogenic candidates. Here we show that mice with partial deficiency of Sentrin/SUMO-specific protease 2 (SENP2) develop spontaneous seizures and sudden death. SENP2 is highly enriched in the hippocampus, often the focus of epileptic seizures. SENP2 deficiency results in hyper-SUMOylation of multiple potassium channels known to regulate neuronal excitability. We demonstrate that the depolarizing M-current conducted by Kv7 channel is significantly diminished in SENP2-deficient hippocampal CA3 neurons, primarily responsible for neuronal hyperexcitability. Following seizures, SENP2-deficient mice develop atrioventricular conduction blocks and cardiac asystole. Both seizures and cardiac conduction blocks can be prevented by retigabine, a Kv7 channel opener. Thus, we uncover a disease-causing role for hyper-SUMOylation in the nervous system and establish an animal model for SUDEP.

Increased spinal cord Na⁺-K⁺-2Cl⁻ cotransporter-1 (NKCC1) activity contributes to impairment of synaptic inhibition in paclitaxel-induced neuropathic pain.

Microtubule-stabilizing agents, such as paclitaxel (Taxol), are effective chemotherapy drugs for treating many cancers, and painful neuropathy is a major dose-limiting adverse effect. Cation-chloride cotransporters, such as Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) and K(+)-Cl(-) cotransporter-2 (KCC2), critically influence spinal synaptic inhibition by regulating intracellular chloride concentrations. Here we show that paclitaxel treatment in rats significantly reduced GABA-induced membrane hyperpolarization and caused a depolarizing shift in GABA reversal potential of dorsal horn neurons. However, paclitaxel had no significant effect on AMPA or NMDA receptor-mediated glutamatergic input from primary afferents to dorsal horn neurons. Paclitaxel treatment significantly increased protein levels, but not mRNA levels, of NKCC1 in spinal cords. Inhibition of NKCC1 with bumetanide reversed the paclitaxel effect on GABA-mediated hyperpolarization and GABA reversal potentials. Also, intrathecal bumetanide significantly attenuated hyperalgesia and allodynia induced by paclitaxel. Co-immunoprecipitation revealed that NKCC1 interacted with ß-tubulin and ß-actin in spinal cords. Remarkably, paclitaxel increased NKCC1 protein levels at the plasma membrane and reduced NKCC1 levels in the cytosol of spinal cords. In contrast, treatment with an actin-stabilizing agent had no significant effect on NKCC1 protein levels in the plasma membrane or cytosolic fractions of spinal cords. In addition, inhibition of the motor protein dynein blocked paclitaxel-induced subcellular redistribution of NKCC1, whereas inhibition of kinesin-5 mimicked the paclitaxel effect. Our findings suggest that increased NKCC1 activity contributes to diminished spinal synaptic inhibition and neuropathic pain caused by paclitaxel. Paclitaxel disrupts intracellular NKCC1 trafficking by interfering with microtubule dynamics and associated motor proteins.

Role of ATP-sensitive potassium channels in modulating nociception in rat model of bone cancer pain.

Bone cancer pain is a major clinical problem and remains difficult to treat. ATP-sensitive potassium (KATP) channels may be involved in regulating nociceptive transmission at the spinal cord level. We determined the role of spinal KATP channels in the control of mechanical hypersensitivity in a rat model of bone cancer pain. The rat model of bone cancer pain was induced by implanting rat mammary gland carcinoma cells (Walker256) into the tibias. KATP modulators (pinacidil and glibenclamide) or the specific Kir6.2-siRNA were injected via an intrathecal catheter. The mechanical withdrawal threshold of rats was tested using von Frey filaments. The Kir6.2 mRNA and protein levels were measured by quantitative PCR and western blots, respectively. Intrathecal injection of pinacidil, a KATP channel opener, significantly increased the tactile withdrawal threshold of cancer cell-injected rats in a dose-dependent manner. In contrast, intrathecal delivery of glibenclamide, a KATP channel blocker, or the specific Kir6.2-siRNA significantly reduced the tactile withdrawal threshold of cancer cell-injected rats. The mRNA and protein levels of Kir6.2 in the spinal cord of cancer cell-injected rats were significantly lower than those in control rats. Our findings suggest that the KATP channel expression level in the spinal cord is reduced in bone cancer pain. Activation of KATP channels at the spinal level reduces pain hypersensitivity associated with bone cancer pain.

Identification of diverse modulators of central and peripheral circadian clocks by high-throughput chemical screening.

The circadian clock coordinates daily oscillations of essential physiological and behavioral processes. Conversely, aberrant clocks with damped amplitude and/or abnormal period have been associated with chronic diseases and aging. To search for small molecules that perturb or enhance circadian rhythms, we conducted a high-throughput screen of approximately 200,000 synthetic compounds using Per2lucSV reporter fibroblast cells and validated 11 independent classes of molecules with Bmal1:luciferase reporter cells as well as with suprachiasmatic nucleus and peripheral tissue explants. Four compounds were found to lengthen the period in both central and peripheral clocks, including three compounds that inhibited casein kinase Iε in vitro and a unique benzodiazepine derivative acting through a non-GABA(A) receptor target. In addition, two compounds acutely induced Per2lucSV reporter bioluminescence, delayed the rhythm, and increased intracellular cAMP levels, but caused rhythm damping. Importantly, five compounds shortened the period of peripheral clocks; among them, four compounds also enhanced the amplitude of central and/or peripheral reporter rhythms. Taken together, these studies highlight diverse activities of drug-like small molecules in manipulating the central and peripheral clocks. These small molecules constitute a toolbox for probing clock regulatory mechanisms and may provide putative lead compounds for treatment of clock-associated diseases.

Electroacupuncture increases CB2 receptor expression on keratinocytes and infiltrating inflammatory cells in inflamed skin tissues of rats.

UNLABELLED: Endogenous cannabinoids and peripheral cannabinoid CB2 receptors (CB2Rs) are involved in the antinociceptive effect of electroacupuncture (EA) on inflammatory pain. However, it remains unclear about how EA affects the expression and distribution patterns of peripheral CB2Rs in inflamed skin tissues. To study this, inflammatory pain was induced by local injection of complete Freund's adjuvant into the hindpaw of rats. The mRNA and protein levels of CB2Rs were quantified by using RTPCR and Western blotting, respectively. The distribution of CB2Rs on keratinocytes and immune cells recruited to the inflamed skin tissues was determined by using double-immunofluorescence labeling. Induction of tissue inflammation significantly increased the mRNA and protein levels of CB2Rs in the skin tissue. Also, both 2 Hz and 100 Hz EA, applied to GB30 and GB34, significantly increased the mRNA and protein levels of CB2Rs in inflamed tissues compared to the sham EA group. CB2Rimmunoreactivities were mainly distributed in keratinocytes, macrophages, and T-lymphocytes in the epidermis and dermis of the inflamed skin tissue. Inflammation caused a significant increase in the number of CB2R-immunoreactive keratinocytes, macrophages, and T-lymphocytes. Furthermore, compared to the sham EA group, EA at 2 or 100 Hz significantly increased the number of keratinocytes, macrophages, and T-lymphocytes with CB2R-immunoreactivity in the inflamed skin tissue. Therefore, our findings suggest that EA is associated with upregulation of local CB2Rs in the inflamed skin tissue. EA primarily potentiates the expression of CB2Rs on keratinocytes and infiltrating inflammatory cells at the site of inflammation. PERSPECTIVE: This study shows that electroacupuncture increases the CB2 receptor expression on keratinocytes and infiltrating inflammatory cells in inflammatory skin tissues. This finding provides new evidence showing the potential role of CB2 receptors in the analgesic effect of acupuncture on inflammatory pain.