Implications of Ape1 in reactive oxygen signaling response following cisplatin treatment of dorsal root ganglion neurons

Peripheral neuropathy is one of the major side-effects of the anticancer drug, cisplatin. Although previous work suggests that this neuropathy correlates with formation of DNA adducts in sensory neurons, growing evidence suggests that cisplatin also increases the generation of reactive oxygen species (ROS), which could cause DNA damage. Apurinic/apyrimidinic endonuclease/redox factor-1 (Ape1/Ref-1) is a multifunctional protein involved in DNA base excision repair (BER) of oxidative DNA damage and in redox regulation of a number of transcription factors. Therefore, we asked whether altering Ape1 functions would influence cisplatin induced neurotoxicity. Sensory neurons in culture were exposed to cisplatin for 24 hrs and several endpoints of toxicity were measured including production of ROS, cell death, apoptosis, and release of the immunoreactive calcitonin gene-related peptide (iCGRP). Reducing expression of Ape1 in neuronal cultures using siRNA enhances cisplatin-induced cell killing, apoptosis, ROS generation and the cisplatin-induced reduction in iCGRP release. Overexpressing wild-type (WT)-Ape1 attenuates all the toxic effects of cisplatin in cells containing normal endogenous levels of Ape1 and in cells with reduced Ape1 levels following Ape1siRNA treatment. Overexpressing the redox deficient/repair competent C65-Ape1 provides partial rescue, while the repair deficient Ape1 (N226A+R177A) does not protect neurons from cisplatin toxicity. We also observe an increase in phosphorylation of p53 following a decrease in Ape1 levels in sensory neuronal cultures. These results strongly support the notion that Ape1 is a potential translational target such that protecting Ape1 levels and particularly its DNA repair function could reduce peripheral neuropathy in patients undergoing cisplatin treatment

Models of inflammation: Carrageenan- or complete Freund's Adjuvant (CFA)-induced edema and hypersensitivity in the rat

Animal models of inflammation are used to assess the production of inflammatory mediators at sites of inflammation, the anti-inflammatory properties of agents such as nonsteroidal anti-inflammatory drugs (NSAIDs), and the efficacy of putative analgesic compounds in reversing cutaneous hypersensitivity. This unit details methods to elicit and measure carrageenan- and complete Freund's adjuvant (CFA)-induced cutaneous inflammation. Due to possible differences between the dorsal root sensory system and the trigeminal sensory system, injections of either the footpad or vibrissal pad are described. In this manner, cutaneous inflammation can be assessed in tissue innervated by the lumbar dorsal root ganglion neurons (footpad) and by the trigeminal ganglion neurons (vibrissal pad)

DNA damage mediates changes in neuronal sensitivity induced by the inflammatory mediators, MCP-1 and LPS, and can be reversed by enhancing the DNA repair function of APE1

Although inflammation-induced peripheral sensitization oftentimes resolves as an injury heals, this sensitization can be pathologically maintained and contribute to chronic inflammatory pain. Numerous inflammatory mediators increase the production of reactive oxygen (ROS) and nitrogen species (RNS) during inflammation and in animal models of chronic neuropathic pain. Our previous studies demonstrate that ROS/RNS and subsequent DNA damage mediate changes in neuronal sensitivity induced by anticancer drugs and by ionizing radiation in sensory neurons, thus we investigated whether inflammation and inflammatory mediators also could cause DNA damage in sensory neurons and whether that DNA damage alters neuronal sensitivity. DNA damage was assessed by pH2A.X expression and the release of the neuropeptide, calcitonin gene-related peptide (CGRP), was measured as an index of neuronal sensitivity. Peripheral inflammation or exposure of cultured sensory neurons to the inflammatory mediators, LPS and MCP-1, elicited DNA damage. Moreover, exposure of sensory neuronal cultures to LPS or MCP-1 resulted in changes in the stimulated release of CGRP, without altering resting release or CGRP content. Genetically enhancing the expression of the DNA repair enzyme, apurinic/apyrimidinic endonuclease (APE1) or treatment with a small-molecule modulator of APE1 DNA repair activity, both which enhance DNA repair, attenuated DNA damage and the changes in neuronal sensitivity elicited by LPS or MCP-1. In conclusion, our studies demonstrate that inflammation or exposure to inflammatory mediators elicits DNA damage in sensory neurons. By enhancing DNA repair, we demonstrate that this DNA damage mediates the alteration of neuronal function induced by inflammatory mediators in peptidergic sensory neurons

The repair function of the multifunctional DNA repair/redox protein APE1 is neuroprotective after ionizing radiation

Although exposure to ionizing radiation (IR) can produce significant neurotoxicity, the mechanisms mediating this toxicity remain to be determined. Previous studies using neurons isolated from the central nervous system show that IR produces reactive oxygen species and oxidative DNA damage in those cells. Because the base excision DNA repair pathway repairs single-base modifications caused by ROS, we asked whether manipulating this pathway by altering APE1 expression would affect radiation-induced neurotoxicity. In cultures of adult hippocampal and sensory neurons, IR produces DNA damage as measured by phosphorylation of histone H2A.X and results in dose-dependent cell death. In isolated sensory neurons, we demonstrate for the first time that radiation decreases the capsaicin-evoked release of the neuropeptide CGRP. Reducing APE1 expression in cultured cells augments IR-induced neurotoxicity, whereas overexpressing APE1 is neuroprotective. Using lentiviral constructs with a neuronal specific promoter that selectively expresses APE1s different functions in neurons, we show that selective expression of the DNA repair competent (redox inactive) APE1 constructs in sensory neurons resurrects cell survival and neuronal function, whereas use of DNA-repair deficient (redox active) constructs is not protective. Use of an APE1 redox-specific inhibitor, APX3330, also facilitates neuronal protection against IR-induced toxicity. These results demonstrate for the first time that the repair function of APE1 is required to protect both hippocampal and DRG neuronal cultures—specifically neuronal cells—from IR-induced damage, while the redox activity of APE1 does not appear to be involved

Models of Inflammation: Carrageenan Air Pouch

The subcutaneous air pouch is an in vivo model that can be used to study the components of acute and chronic inflammation, the resolution of the inflammatory response, the oxidative stress response, and potential therapeutic targets for treating inflammation. Injection of irritants into an air pouch in rats or mice induces an inflammatory response that can be quantified by the volume of exudate produced, the infiltration of cells, and the release of inflammatory mediators. The model presented in this unit has been extensively used to identify potential anti-inflammatory drugs

Long-term exposure to PGE2 causes homologous desensitization of receptor-mediated activation of protein kinase A

BACKGROUND: Acute exposure to prostaglandin E2 (PGE2) activates EP receptors in sensory neurons which triggers the cAMP-dependent protein kinase A (PKA) signaling cascade resulting in enhanced excitability of the neurons. With long-term exposure to PGE2, however, the activation of PKA does not appear to mediate persistent PGE2-induced sensitization. Consequently, we examined whether homologous desensitization of PGE2-mediated PKA activation occurs after long-term exposure of isolated sensory neurons to the eicosanoid. METHODS: Sensory neuronal cultures were harvested from the dorsal root ganglia of adult male Sprague-Dawley rats. The cultures were pretreated with vehicle or PGE2 and used to examine signaling mechanisms mediating acute versus persistent sensitization by exposure to the eicosanoid using enhanced capsaicin-evoked release of immunoreactive calcitonin gene-related peptide (iCGRP) as an endpoint. Neuronal cultures chronically exposed to vehicle or PGE2 also were used to study the ability of the eicosanoid and other agonists to activate PKA and whether long-term exposure to the prostanoid alters expression of EP receptor subtypes. RESULTS: Acute exposure to 1 μM PGE2 augments the capsaicin-evoked release of iCGRP, and this effect is blocked by the PKA inhibitor H-89. After 5 days of exposure to 1 μM PGE2, administration of the eicosanoid still augments evoked release of iCGRP, but the effect is not attenuated by inhibition of PKA or by inhibition of PI3 kinases. The sensitizing actions of PGE2 after acute and long-term exposure were attenuated by EP2, EP3, and EP4 receptor antagonists, but not by an EP1 antagonist. Exposing neuronal cultures to 1 μM PGE2 for 12 h to 5 days blocks the ability of PGE2 to activate PKA. The offset of the desensitization occurs within 24 h of removal of PGE2 from the cultures. Long-term exposure to PGE2 also results in desensitization of the ability of a selective EP4 receptor agonist, L902688 to activate PKA, but does not alter the ability of cholera toxin, forskolin, or a stable analog of prostacyclin to activate PKA. CONCLUSIONS: Long-term exposure to PGE2 results in homologous desensitization of EP4 receptor activation of PKA, but not to neuronal sensitization suggesting that activation of PKA does not mediate PGE2-induced sensitization after chronic exposure to the eicosanoid

Mechanisms of the release of anterogradely transported neurotrophin-3 from axon terminals

Neurotrophins have profound effects on synaptic function and structure. They can be derived from presynaptic, as well as postsynaptic, sites. To date, it has not been possible to measure the release of neurotrophins from axon terminals in intact tissue. We implemented a novel, extremely sensitive assay for the release and transfer of anterogradely transported neurotrophin-3 (NT-3) from a presynaptic to a postsynaptic location that uses synaptosomal fractionation after introduction of radiolabeled NT-3 into the retinotectal projection of chick embryos. Release of the anterogradely transported NT-3 in intact tissue was assessed by measuring the amount remaining in synaptosomal preparations after treatment of whole tecta with pharmacological agents. Use of this assay reveals that release of NT-3 from axon terminals is increased by depolarization, calcium influx via N-type calcium channels, and cAMP analogs, and release is most profoundly increased by excitation with kainic acid or mobilization of calcium from intracellular stores. NT-3 release depends on extracellular sodium, CaM kinase II activity, and requires intact microtubules and microfilaments. Dantrolene inhibits the high potassium-induced release of NT-3, indicating that release of calcium from intracellular stores is required. Tetanus toxin also inhibits NT-3 release, suggesting that intact synaptobrevin or synaptobrevin-like molecules are required for exocytosis. Ultrastructural autoradiography and immunolabel indicate that NT-3 is packaged in presumptive large dense-core vesicles. These data show that release of NT-3 from axon terminals depends on multiple regulatory proteins and ions, including the mobilization of local calcium. The data provide insight in the mechanisms of anterograde neurotrophins as synaptic modulators

Vasodilatation in the rat dorsal hindpaw induced by activation of sensory neurons is reduced by Paclitaxel

Peripheral neuropathy is a major side effect following treatment with the cancer chemotherapeutic drug paclitaxel. Whether paclitaxel-induced peripheral neuropathy is secondary to altered function of small diameter sensory neurons remains controversial. To ascertain whether the function of the small diameter sensory neurons was altered following systemic administration of paclitaxel, we injected male Sprague Dawley rats with 1 mg/kg paclitaxel every other day for a total of four doses and examined vasodilatation in the hindpaw at day 14 as an indirect measure of calcitonin gene related peptide (CGRP) release. In paclitaxel-treated rats, the vasodilatation induced by either intradermal injection of capsaicin into the hindpaw or electrical stimulation of the sciatic nerve was significantly attenuated in comparison to vehicle-injected animals. Paclitaxel treatment, however, did not affect direct vasodilatation induced by intradermal injection of methacholine or CGRP, demonstrating that the blood vessels’ ability to dilate was intact. Paclitaxel treatment did not alter the compound action potentials or conduction velocity of C-fibers. The stimulated release of CGRP from the central terminals in the spinal cord was not altered in paclitaxel-injected animals. These results suggest that paclitaxel affects the peripheral endings of sensory neurons to alter transmitter release, and this may contribute to the symptoms seen in neuropathy

Paclitaxel alters the evoked release of calcitonin gene-related peptide from rat sensory neurons in culture

Peripheral neuropathy (PN) is a debilitating and dose-limiting side effect of treatment with the chemotherapeutic agent, paclitaxel. Understanding the effects of paclitaxel on sensory neuronal function and the signaling pathways which mediate these paclitaxel-induced changes in function are critical for the development of therapies to prevent or alleviate the PN. The effects of long-term administration of paclitaxel on the function of sensory neurons grown in culture, using the release of the neuropeptide calcitonin gene-related peptide (CGRP) as an endpoint of sensory neuronal function, were examined. Dorsal root ganglion cultures were treated with low (10 nM) and high (300 nM) concentrations of paclitaxel for 1, 3, or 5 days. Following paclitaxel treatment, the release of CGRP was determined using capsaicin, a TRPV1 agonist; allyl isothiocyanate (AITC), a TRPA1 agonist; or high extracellular potassium. The effects of paclitaxel on the release of CGRP were stimulant-, concentration-, and time-dependent. When neurons were stimulated with capsaicin or AITC, a low concentration of paclitaxel (10nM) augmented transmitter release, whereas a high concentration (300 nM) reduced transmitter release in a time-dependent manner; however, when high extracellular potassium was used as the evoking stimulus, all concentrations of paclitaxel augmented CGRP release from sensory neurons. These results suggest that paclitaxel alters the function of sensory neurons in vitro, and suggest that the mechanisms by which paclitaxel alters neuronal function may include functional changes in TRP channel activity. The described in vitro model will facilitate future studies to identify the signaling pathways by which paclitaxel alters neuronal sensitivity

Role of APE1 in differentiated neuroblastoma SH-SY5Y cells in response to oxidative stress; Use of APE1 small molecule inhibitors to delineate APE1 functions

Oxidative DNA damage has been implicated in a number of central nervous system pathologies. The base excision repair (BER) pathway is one of the most important cellular protection mechanisms that respond to oxidative DNA damage. Human apurinic (apyrimidinic) endonuclease/redox effector factor (APE1/Ref-1 or APE1) is an essential enzyme in the BER pathway and is expressed in both mitotic and post-mitotic cells in humans. In neurons, a reduction of APE1 expression increases chemotherapy-induced cytotoxicity, while overexpression of APE1 protects cells against the cytotoxicity. However, given the multiple functions of APE1, knockdown of total APE1 is not completely informative of whether it is the redox or DNA repair activity, or interactions with other proteins. Therefore, the use of selective small molecules that can block each function independent of the other is of great benefit in ascertaining APE1 function in post-mitotic cells. In this study, we chose differentiated SH-SY5Y cells as our post-mitotic cell line model to investigate whether a drug-induced decrease in APE1 DNA repair or redox activity contributes to the growth and survival of post-mitotic cells under oxidative DNA damaging conditions. Here, we demonstrate that overexpression of WT-APE1 or C65-APE1 (repair competent) results in significant increase in cell viability after exposure to H2O2. However, the 177/226-APE1 (repair deficient) did not show a protective effect. This phenomenon was further confirmed by the use of methoxyamine (MX), which blocks the repair activity of APE1 that results in enhanced cell killing and apoptosis in differentiated SH-SY5Y cells and in neuronal cultures after oxidative DNA damaging treatments. Blocking APE1 redox function by a small molecule inhibitor, BQP did not decrease viability of SH-SY5Y cells or neuronal cultures following oxidative DNA damaging treatments. Our results demonstrate that the DNA repair function of APE1 contributes to the survival of nondividing post-mitotic cells following oxidative DNA damage