Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

United States Army Medical Research Institute of Chemical Defense

Nerve agent-induced seizures cause neuronal damage in brain limbic and cortical circuits leading to persistent behavioral and cognitive deficits. Without aggressive anticholinergic and benzodiazepine therapy, seizures can be prolonged and neuronal damage progresses for extended periods of time. The objective of this study was to determine the effects of the nerve agent soman on expression of cyclooxygenase-2 (COX-2), the initial enzyme in the biosynthetic pathway of the proinflammatory prostaglandins and a factor that has been implicated in seizure initiation and propagation. Rats were exposed to a toxic dose of soman and scored behaviorally for seizure intensity. Expression of COX-2 was determined throughout brain from 4h to 7 days after exposure by immunohistochemistry and immunoblotting. Microglial activation and astrogliosis were assessed microscopically over the same time-course. Soman increased COX-2 expression in brain regions known to be damaged by nerve agents (e.g., hippocampus, amygdala, piriform cortex and thalamus). COX-2 expression was induced in neurons, and not in microglia or astrocytes, and remained elevated through 7 days. The magnitude of COX-2 induction was correlated with seizure intensity. COX-1 expression was not changed by soman. Increased expression of neuronal COX-2 by soman is a late-developing response relative to other signs of acute physiological distress caused by nerve agents. COX-2-mediated production of prostaglandins is a consequence of the seizure-induced neuronal damage, even after survival of the initial cholinergic crisis is assured. COX-2 inhibitors should be considered as adjunct therapy in nerve agent poisoning to minimize nerve agent-induced seizure activity.

Soman increases neuronal COX-2 levels: possible link between seizures and protracted neuronal damage.

Nerve agents are deadly threats to military and civilian populations around the world. Nerve agents cause toxicity to peripheral and central sites through the irreversible inhibition of acetylcholinesterase, the enzyme that metabolizes acetylcholine. Excessive acetylcholine accumulation in synapses results in status epilepticus in the central nervous system. Prolonged status epilepticus leads to brain damage, neurological dysfunction and poor outcome. Anticonvulsants are effective but must be given rapidly following exposure. Because these agents cause mass casualties, effective neuroprotective agents are needed to reduce brain damage and improve cognitive outcome. α-Linolenic acid is an omega-3 fatty acid that is found in vegetable products and has no known side effects. α-Linolenic acid is neuroprotective against kainic acid-induced brain damage in vivo, but its neuroprotective efficacy against nerve agents is unknown. α-Linolenic acid also exerts anti-depressant and anti-inflammatory activities and enhances synaptic plasticity in vivo. These properties make this polyunsaturated fatty acid (PUFA) a potential candidate against nerve agent-induced neuropathology. Here we show that α-linolenic acid is neuroprotective against soman-induced neuropathology in either a pretreatment or post-treatment paradigm. We also show that subcutaneous injection of α-linolenic acid shows greater neuroprotective efficacy compared with intravenous injection in a brain region-specific manner.

Alpha-linolenic acid is a potent neuroprotective agent against soman-induced neuropathology.

Simultaneous use of cell-permeant and impermeant fluorescent nuclear dyes is a common method to study cell viability and cell death progression. Although these assays are usually conducted as end-point studies, time-lapse imaging offers a powerful technique to distinguish temporal changes in cell viability at single-cell resolution. SYTO 13 and Hoechst 33342 are two commonly used cell-permeant nuclear dyes; however their suitability for live imaging has not been well characterized. We compare end-point assays with time-lapse imaging studies over a 6 h period to evaluate the compatibility of these two dyes with longitudinal imaging, using both control neurons and an apoptotic neuron model.

Compatibility of SYTO 13 and Hoechst 33342 for longitudinal imaging of neuron viability and cell death.

A subset of victims of ocular sulfur mustard (SM) exposure develops an irreversible, idiotypic keratitis with associated secondary pathologies, collectively referred to as mustard gas keratopathy (MGK). MGK involves a progressive corneal degeneration resulting in chronic ocular discomfort and impaired vision for which clinical interventions have typically had poor outcomes. Using a rabbit corneal vapor exposure model, we previously demonstrated a clinical progression with acute and chronic sequelae similar to that observed in human casualties. However, a better understanding of the temporal changes that occur during the biphasic SM injury is crucial to mechanistic understanding and therapeutic development. Here we evaluate the histopathologic, biochemical and ultrastructural expressions of pathogenesis of the chronic SM injury over eight weeks. We confirm that MGK onset exhibits a biphasic trajectory involving corneal surface regeneration over the first two weeks, followed by the rapid development and progressive degeneration of corneal structure. Preclinical markers of corneal dysfunction were identified, including destabilization of the basal corneal epithelium, basement membrane zone abnormalities and stromal deformation. Clinical sequelae of MGK appeared abruptly three weeks after exposure, and included profound anterior edema, recurring corneal erosions, basement membrane disorganization, basal cell necrosis and stromal degeneration. Unlike resolved corneas, MGK corneas exhibited frustrated corneal wound repair, with significantly elevated histopathology scores. Increased lacrimation, disruption of the basement membrane and accumulation of pro-inflammatory mediators in the aqueous humor provide several mechanisms for corneal degeneration. These data suggest that the chronic injury is fundamentally distinct from the acute lesion, involving injury mechanisms that operate on different time scales and in different corneal tissues. Corneal edema appears to be the principal pathology of MGK, in part resulting from persistent necrosis of the basal corneal epithelium and deterioration of the basement membrane. The findings also provide a potential explanation as to why administration of anti-inflammatories transiently delays, but does not prevent, the development of MGK sequelae.

Architectural and biochemical expressions of mustard gas keratopathy: preclinical indicators and pathogenic mechanisms.

Recently, there has been a strong emphasis on identifying an in vitro model for neurotoxicity research that combines the biological relevance of primary neurons with the scalability, reproducibility and genetic tractability of continuous cell lines. Derived neurons should be homotypic, exhibit neuron-specific gene expression and morphology, form functioning synapses and consistently respond to neurotoxins in a fashion indistinguishable from primary neurons. However, efficient methods to produce neuronal populations that are suitable alternatives to primary neurons have not been available.

High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research.

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.

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

Sulfur mustard (SM) is a highly reactive vesicant that causes severe ocular injuries. Following exposure to moderate or high doses, a subset of victims develops a chronic injury known as mustard gas keratopathy (MGK) involving a keratitis of unknown etiopathogenesis with secondary keratopathies such as persistent epithelial lesions, corneal neovascularization, and progressive corneal degeneration. This study was designed to determine whether SM exposure evokes acute endothelial toxicity and to determine whether endothelial pathologies were specifically observed in MGK corneas as opposed to healed corneas.

Structural, morphological, and functional correlates of corneal endothelial toxicity following corneal exposure to sulfur mustard vapor.

Using paired-end RNA sequencing, we have quantified the deep transcriptional changes that occur during differentiation of murine embryonic stem cells into a highly enriched population of glutamatergic cortical neurons. These data provide a detailed and nuanced account of longitudinal changes in the transcriptome during neurogenesis and neuronal maturation, starting from mouse embryonic stem cells and progressing through neuroepithelial stem cell induction, radial glial cell formation, neurogenesis, neuronal maturation and cortical patterning. Understanding the transcriptional mechanisms underlying the differentiation of stem cells into mature, glutamatergic neurons of cortical identity has myriad applications, including the elucidation of mechanisms of cortical patterning; identification of neurogenic processes; modeling of disease states; detailing of the host cell response to neurotoxic stimuli; and determination of potential therapeutic targets. In future work we anticipate correlating changes in longitudinal gene expression to other cell parameters, including neuronal function as well as characterizations of the proteome and metabolome. In this data article, we describe the methods used to produce the data and present the raw sequence read data in FASTQ files, sequencing run statistics and a summary flatfile of raw counts for 22,164 genes across 31 samples, representing 3-5 biological replicates at each timepoint. We propose that this data will be a valuable contribution to diverse research efforts in bioinformatics, stem cell research and developmental neuroscience studies.

Megan Lyman

Research Division,
Cellular Molecular Biology Branch,
Research Division, Cellular Molecular Biology Branch

United States Army Medical Research Institute of Chemical Defense

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Megan Lyman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Research Division,Cellular Molecular Biology Branch,Research Division, Cellular Molecular Biology Branch

United States Army Medical Research Institute of Chemical Defense

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Research Division,
Cellular Molecular Biology Branch,
Research Division, Cellular Molecular Biology Branch