Granulocyte Colony-Stimulating Factor (Neupogen®; Filgrastim) Accelerates Neutrophil Recovery in a Rodent Model of Sulfur Mustard-Induced Hematologic Toxicity.

OBJECTIVE: Evidence of myelosuppression has been negatively correlated with patient outcomes following cases of high dose sulfur mustard (SM) exposure. These hematologic complications can negatively impact overall immune function and increase the risk of infection and life-threatening septicemia. Currently, there are no approved medical treatments for the myelosuppressive effects of SM exposure. METHODS: Leveraging a recently developed rodent model of SM-induced hematologic toxicity, post-exposure efficacy testing of the granulocyte colony-stimulating factor drug Neupogen® was performed in rats intravenously challenged with SM. Before efficacy testing, pharmacokinetic/pharmacodynamic analyses were performed in naïve rats to identify the apparent human equivalent dose of Neupogen® for efficacy evaluation. RESULTS: When administered 1 d after SM-exposure, daily subcutaneous Neupogen® treatment did not prevent the delayed onset of hematologic toxicity but significantly accelerated recovery from neutropenia. Compared with SM controls, Neupogen®-treated animals recovered body weight faster, resolved toxic clinical signs more rapidly, and did not display transient febrility at time points generally concurrent with marked pancytopenia. CONCLUSIONS: Collectively, this work corroborates the results of a previous pilot large animal study, validates the utility of a rodent screening model, and provides further evidence for the potential clinical utility of Neupogen® as an adjunct treatment following SM exposure.

A Rodent Model of Sulfur Mustard Hematologic Toxicity for the Efficacy Evaluation of Candidate Medical Countermeasures.

INTRODUCTION: While exposure to sulfur mustard (SM) is commonly associated with the production of vesicating dermal, ocular, and respiratory injuries, systemic damage to bone marrow and lymphatic tissue can decrease critical immune cell populations leading to higher susceptibility to life-threatening infection and septicemia. There are currently no approved medical countermeasures for SM-induced myelosuppression. An intravenous SM challenge model was developed in adult rats as a preliminary proof-of-principle platform to evaluate the efficacy of candidate immunostimulants. MATERIALS AND METHODS: Adult male and female Sprague Dawley rats were exposed to SM through tail vein injection. Toxicity progression was monitored through clinical observations, body weights, body temperatures, hematology, serum clinical chemistry, and flow cytometry of blood and bone marrow samples. RESULTS: Following SM exposure, overt toxicity progression was characterized by weight loss, changes in body temperature, and manifestation of toxic clinical signs (diarrhea, lethargy, hunched posture, rough hair coat, respiratory distress, and death). Drastic alterations in complete blood cell profiles included an early-onset lymphopenia followed by a delayed-onset neutropenia and thrombocytopenia. Only transient changes in serum clinical chemistry parameters were observed. Flow cytometry analysis of circulating blood revealed that B-cells were more predominantly affected by SM exposure than T-cells. Challenge with SM resulted in loss of hematopoietic and mesenchymal stem cell populations in the bone marrow. CONCLUSIONS: The small animal model developed in this study replicates many key aspects of human SM exposures and should serve as a relevant, rapid, and cost-effective platform to screen candidate medical countermeasures for SM-induced hematologic toxicity.

Liposomal Encapsulated FSC231, a PICK1 Inhibitor, Prevents the Ischemia/Reperfusion-Induced Degradation of GluA2-Containing AMPA Receptors.

Strokes remain one of the leading causes of disability within the United States. Despite an enormous amount of research effort within the scientific community, very few therapeutics are available for stroke patients. Cytotoxic accumulation of intracellular calcium is a well-studied phenomenon that occurs following ischemic stroke. This intracellular calcium overload results from excessive release of the excitatory neurotransmitter glutamate, a process known as excitotoxicity. Calcium-permeable AMPA receptors (AMPARs), lacking the GluA2 subunit, contribute to calcium cytotoxicity and subsequent neuronal death. The internalization and subsequent degradation of GluA2 AMPAR subunits following oxygen-glucose deprivation/reperfusion (OGD/R) is, at least in part, mediated by protein-interacting with C kinase-1 (PICK1). The purpose of the present study is to evaluate whether treatment with a PICK1 inhibitor, FSC231, prevents the OGD/R-induced degradation of the GluA2 AMPAR subunit. Utilizing an acute rodent hippocampal slice model system, we determined that pretreatment with FSC231 prevented the OGD/R-induced association of PICK1-GluA2. FSC231 treatment during OGD/R rescues total GluA2 AMPAR subunit protein levels. This suggests that the interaction between GluA2 and PICK1 serves as an important step in the ischemic/reperfusion-induced reduction in total GluA2 levels.

Characterization and treatment of spontaneous recurrent seizures following nerve agent-induced status epilepticus in mice.

PURPOSE: To develop and characterize a mouse model of spontaneous recurrent seizures following nerve agent-induced status epilepticus (SE) and test the efficacy of existing antiepileptic drugs. METHODS: SE was induced in telemeterized male C57Bl6/J mice by soman exposure, and electroencephalographic activity was recorded for 4-6 weeks. Mice were treated with antiepileptic drugs (levetiracetam, valproic acid, phenobarbital) or corresponding vehicles for 14 d after exposure, followed by 14 d of drug washout. Survival, body weight, seizure characteristics, and histopathology were used to characterize the acute and chronic effects of nerve agent exposure and to evaluate the efficacy of treatments in mitigating or preventing neurological effects. RESULTS: Spontaneous recurrent seizures manifested in all survivors, but the number and frequency of seizures varied considerably among mice. In untreated mice, seizures became longer over time. Moderate to severe histopathology was observed in the amygdala, piriform cortex, and CA1. Levetiracetam provided modest improvements in neurological parameters such as reduced spike rate and improved histopathology scores, whereas valproic acid and phenobarbital were largely ineffective. CONCLUSIONS: This model of post-SE spontaneous recurrent seizures differs from other experimental models in the brief latency to seizure development, the occurrence of seizures in 100 % of exposed animals, and the lack of damage to CA4/dentate gyrus. It may serve as a useful tool for rapidly and efficiently screening novel therapies that would be effective against severe epilepsy cases.

In vitro and ex vivo screening of candidate therapeutics to restore neurotransmission in nerve terminals intoxicated by botulinum neurotoxin serotype A1.

Botulinum neurotoxins (BoNTs) are exceedingly potent neurological poisons that block cholinergic release in the peripheral nervous system and cause death by asphyxiation. While post-exposure prophylaxis can effectively eliminate toxin in the bloodstream, there are no clinically effective treatments to prevent or reverse disease once BoNT has entered the neuron. To address the need for post-symptomatic countermeasures, we designed and developed an in vitro assay based on whole-cell, patch-clamp electrophysiological monitoring of miniature excitatory post-synaptic currents in synaptically active murine embryonic stem cell-derived neurons. This synaptic function-based assay was used to assess the efficacy of rationally selected drugs to restore neurotransmission in neurons comprehensively intoxicated by BoNT/A. Based on clinical reports suggesting that elevated Ca2+ signaling promotes symptomatic relief from botulism, we identified seven candidate drugs that modulate presynaptic Ca2+ signaling and assessed their ability to reverse BoNT/A-induced synaptic blockade. The most effective drugs from the screen were found to phasically agonize voltage-gated calcium channel (VGCC) activity. Lead candidates were then applied to ex vivo studies in BoNT/A-paralyzing mouse phrenic nerve-hemidiaphragm (PND) preparations. Treatment of PNDs with VGCC agonists after paralytic onset transiently potentiated nerve-elicited muscle contraction and delayed progression to neuromuscular failure. Collectively, this study suggests that Ca2+-modulating drugs represent a novel symptomatic treatment for neuromuscular paralysis following BoNT/A poisoning.

Use-dependent potentiation of voltage-gated calcium channels rescues neurotransmission in nerve terminals intoxicated by botulinum neurotoxin serotype A.

Botulinum neurotoxins (BoNTs) are highly potent toxins that cleave neuronal SNARE proteins required for neurotransmission, causing flaccid paralysis and death by asphyxiation. Currently, there are no clinical treatments to delay or reverse BoNT-induced blockade of neuromuscular transmission. While aminopyridines have demonstrated varying efficacy in transiently reducing paralysis following BoNT poisoning, the precise mechanisms by which aminopyridines symptomatically treat botulism are not understood. Here we found that activity-dependent potentiation of presynaptic voltage-gated calcium channels (VGCCs) underlies 3,4-diaminopyridine (3,4-DAP)-mediated rescue of neurotransmission in central nervous system synapses and mouse diaphragm neuromuscular junctions fully intoxicated by BoNT serotype A. Combinatorial treatments with 3,4-DAP and VGCC agonists proved synergistic in restoring suprathreshold endplate potentials in mouse diaphragms fully intoxicated by BoNT/A. In contrast, synapses fully intoxicated by BoNT serotypes D or E were refractory to synaptic rescue by any treatment. We interpret these data to propose that increasing the duration or extent of VGCC activation prolongs the opportunity for low-efficiency fusion by fusogenic complexes incorporating BoNT/A-cleaved SNAP-25. The identification of VGCC agonists that rescue neurotransmission in BoNT/A-intoxicated synapses provides compelling evidence for potential therapeutic utility in some cases of human botulism.

Engineering Botulinum Neurotoxin C1 as a Molecular Vehicle for Intra-Neuronal Drug Delivery.

Botulinum neurotoxin (BoNT) binds to and internalizes its light chain into presynaptic compartments with exquisite specificity. While the native toxin is extremely lethal, bioengineering of BoNT has the potential to eliminate toxicity without disrupting neuron-specific targeting, thereby creating a molecular vehicle capable of delivering therapeutic cargo into the neuronal cytosol. Building upon previous work, we have developed an atoxic derivative (ad) of BoNT/C1 through rationally designed amino acid substitutions in the metalloprotease domain of wild type (wt) BoNT/C1. To test if BoNT/C1 ad retains neuron-specific targeting without concomitant toxic host responses, we evaluated the localization, activity, and toxicity of BoNT/C1 ad in vitro and in vivo. In neuronal cultures, BoNT/C1 ad light chain is rapidly internalized into presynaptic compartments, but does not cleave SNARE proteins nor impair spontaneous neurotransmitter release. In mice, systemic administration resulted in the specific co-localization of BoNT/C1 ad with diaphragmatic motor nerve terminals. The mouse LD50 of BoNT/C1 ad is 5 mg/kg, with transient neurological symptoms emerging at sub-lethal doses. Given the low toxicity and highly specific neuron-targeting properties of BoNT/C1 ad, these data suggest that BoNT/C1 ad can be useful as a molecular vehicle for drug delivery to the neuronal cytoplasm.

Botulinum and Tetanus Neurotoxin-Induced Blockade of Synaptic Transmission in Networked Cultures of Human and Rodent Neurons.

Clinical manifestations of tetanus and botulism result from an intricate series of interactions between clostridial neurotoxins (CNTs) and nerve terminal proteins that ultimately cause proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and functional blockade of neurotransmitter release. Although detection of cleaved SNARE proteins is routinely used as a molecular readout of CNT intoxication in cultured cells, impaired synaptic function is the pathophysiological basis of clinical disease. Work in our laboratory has suggested that the blockade of synaptic neurotransmission in networked neuron cultures offers a phenotypic readout of CNT intoxication that more closely replicates the functional endpoint of clinical disease. Here, we explore the value of measuring spontaneous neurotransmission frequencies as novel and functionally relevant readouts of CNT intoxication. The generalizability of this approach was confirmed in primary neuron cultures as well as human and mouse stem cell-derived neurons exposed to botulinum neurotoxin serotypes A-G and tetanus neurotoxin. The sensitivity and specificity of synaptic activity as a reporter of intoxication was evaluated in assays representing the principal clinical and research purposes of in vivo studies. Our findings confirm that synaptic activity offers a novel and functionally relevant readout for the in vitro characterizations of CNTs. They further suggest that the analysis of synaptic activity in neuronal cell cultures can serve as a surrogate for neuromuscular paralysis in the mouse lethal assay, and therefore is expected to significantly reduce the need for terminal animal use in toxin studies and facilitate identification of candidate therapeutics in cell-based screening assays.

Accelerated intoxication of GABAergic synapses by botulinum neurotoxin A disinhibits stem cell-derived neuron networks prior to network silencing.

Botulinum neurotoxins (BoNTs) are extremely potent toxins that specifically cleave SNARE proteins in peripheral synapses, preventing neurotransmitter release. Neuronal responses to BoNT intoxication are traditionally studied by quantifying SNARE protein cleavage in vitro or monitoring physiological paralysis in vivo. Consequently, the dynamic effects of intoxication on synaptic behaviors are not well-understood. We have reported that mouse embryonic stem cell-derived neurons (ESNs) are highly sensitive to BoNT based on molecular readouts of intoxication. Here we study the time-dependent changes in synapse- and network-level behaviors following addition of BoNT/A to spontaneously active networks of glutamatergic and GABAergic ESNs. Whole-cell patch-clamp recordings indicated that BoNT/A rapidly blocked synaptic neurotransmission, confirming that ESNs replicate the functional pathophysiology responsible for clinical botulism. Quantitation of spontaneous neurotransmission in pharmacologically isolated synapses revealed accelerated silencing of GABAergic synapses compared to glutamatergic synapses, which was consistent with the selective accumulation of cleaved SNAP-25 at GAD1(+) pre-synaptic terminals at early timepoints. Different latencies of intoxication resulted in complex network responses to BoNT/A addition, involving rapid disinhibition of stochastic firing followed by network silencing. Synaptic activity was found to be highly sensitive to SNAP-25 cleavage, reflecting the functional consequences of the localized cleavage of the small subpopulation of SNAP-25 that is engaged in neurotransmitter release in the nerve terminal. Collectively these findings illustrate that use of synaptic function assays in networked neurons cultures offers a novel and highly sensitive approach for mechanistic studies of toxin:neuron interactions and synaptic responses to BoNT.

Identification of NADPH oxidase as a key mediator in the post-ischemia-induced sequestration and degradation of the GluA2 AMPA receptor subunit.

A hallmark of ischemic/reperfusion injury is a change in subunit composition of synaptic 2-amino-3-(3-hydroxy-5-methylisoazol-4-yl)propionic acid receptors (AMPARs). This change in AMPAR subunit composition leads to an increase in surface expression of GluA2-lacking Ca(2+) /Zn(2+) permeable AMPARs. These GluA2-lacking AMPARs play a key role in promoting delayed neuronal death following ischemic injury. At present, the mechanism(s) responsible for the ischemia/reperfusion-induced subunit composition switch and degradation of the GluA2 subunit remain unclear. In this study, we investigated the role of NADPH oxidase, and its importance in mediating endocytosis and subsequent degradation of the GluA2 AMPAR subunit in adult rat hippocampal slices subjected to oxygen-glucose deprivation/reperfusion (OGD/R) injury. In hippocampal slices pre-treated with the NADPH oxidase inhibitor apocynin attenuated OGD/R-mediated sequestration of GluA2 and GluA1 as well as prevent the degradation of GluA2. We provide compelling evidence that NADPH oxidase mediated sequestration of GluA1- and GluA2- involved activation of p38 MAPK. Furthermore, we demonstrate that inhibition of NADPH oxidase blunts the OGD/R-induced association of GluA2 with protein interacting with C kinase-1. In summary, this study identifies a novel mechanism that may underlie the ischemia/reperfusion-induced AMPAR subunit composition switch and a potential therapeutic target.

Novel application of stem cell-derived neurons to evaluate the time- and dose-dependent progression of excitotoxic injury.

Glutamate receptor (GluR)-mediated neurotoxicity is implicated in a variety of disorders ranging from ischemia to neural degeneration. Under conditions of elevated glutamate, the excessive activation of GluRs causes internalization of pathologic levels of Ca(2+), culminating in bioenergetic failure, organelle degradation, and cell death. Efforts to characterize cellular and molecular aspects of excitotoxicity and conduct therapeutic screening for pharmacologic inhibitors of excitogenic progression have been hindered by limitations associated with primary neuron culture. To address this, we evaluated glutamate-induced neurotoxicity in highly enriched glutamatergic neurons (ESNs) derived from murine embryonic stem cells. As of 18 days in vitro (DIV 18), ESNs were synaptically coupled, exhibited spontaneous network activity with neurotypic mEPSCs and expressed NMDARs and AMPARs with physiological current:voltage behaviors. Addition of 0.78-200 µM glutamate evoked reproducible time- and dose-dependent metabolic failure in 6 h, with a calculated EC50 value of 0.44 µM at 24 h. Using a combination of cell viability assays and electrophysiology, we determined that glutamate-induced toxicity was specifically mediated by NMDARs and could be inhibited by addition of NMDAR antagonists, increased extracellular Mg(2+) or substitution of Ba(2+) for Ca(2+). Glutamate treatment evoked neurite fragmentation and focal swelling by both immunocytochemistry and scanning electron microscopy. Presentation of morphological markers of cell death was dose-dependent, with 0.78-200 µM glutamate resulting in apoptosis and 3000 µM glutamate generating a mixture of necrosis and apoptosis. Addition of neuroprotective small molecules reduced glutamate-induced neurotoxicity in a dose-dependent fashion. These data indicate that ESNs replicate many of the excitogenic mechanisms observed in primary neuron culture, offering a moderate-throughput model of excitotoxicity that combines the verisimilitude of primary neurons with the flexibility and scalability of cultured cells. ESNs therefore offer a physiologically relevant platform that exhibits characteristic NMDAR responses, and appears suitable to evaluate molecular mechanisms of glutamate-induced excitotoxicity and screen for candidate therapeutics.

NADPH oxidase mediates the oxygen-glucose deprivation/reperfusion-induced increase in the tyrosine phosphorylation of the N-methyl-D-aspartate receptor NR2A subunit in retinoic acid differentiated SH-SY5Y Cells.

BACKGROUND: Evidence exists that oxidative stress promotes the tyrosine phosphorylation of N-methyl-D-aspartate receptor (NMDAR) subunits during post-ischemic reperfusion of brain tissue. Increased tyrosine phosphorylation of NMDAR NR2A subunits has been reported to potentiate receptor function and exacerbate NMDAR-induced excitotoxicity. Though the effect of ischemia on tyrosine phosphorylation of NMDAR subunits has been well documented, the oxidative stress signaling cascades mediating the enhanced tyrosine phosphorylation of NR2A subunits remain unclear. RESULTS: We report that the reactive oxygen species (ROS) generator NADPH oxidase mediates an oxidative stress-signaling cascade involved in the increased tyrosine phosphorylation of the NR2A subunit in post-ischemic differentiated SH-SY5Y neuroblastoma cells. Inhibition of NADPH oxidase attenuated the increased tyrosine phosphorylation of the NMDAR NR2A subunit, while inhibition of ROS production from mitochondrial or xanthine oxidase sources failed to dampen the post-ischemic increase in tyrosine phosphorylation of the NR2A subunit. Additionally, inhibition of NADPH oxidase blunted the interaction of activated Src Family Kinases (SFKs) with PSD-95 induced by ischemia/reperfusion. Lastly, inhibition of NADPH oxidase also markedly reduced cell death in post-ischemic SH-SY5Y cells stimulated by NMDA. CONCLUSIONS: These data indicate that NADPH oxidase has a key role in facilitating NMDAR NR2A tyrosine phosphorylation via SFK activation during post-ischemic reperfusion.