U.S. Army Medical Research Institute of Chemical Defense, Comparative Medicine Division
||Aberdeen Proving Ground, MD 210105425
Our lab currently comprises 15 personnel, including 5 postdoctoral fellows, 3 civilians, and 7 technicians. We conduct research in three areas: acute and delayed treatments for nerve agent-induced seizure activity, treatments to reverse botulinum neurotoxin-induced paralysis, and understanding the acute and chronic pathophysiologies involved in ocular sulfur mustard injury. These studies utilize diverse models, including biochemical assays, primary neuron culture, ex vivo electrophysiology, whole-cell patch-clamp electrophysiology, cortical electroencephalograms, diaphragmatic electromyograms, respiratory function assays and whole animal survival and behavioral assessments. We are seeking highly motivated and technically proficient postdoctoral fellows to support innovative studies in the following research areas:
(1) Biological neurotoxin countermeasures. The seven Clostridium botulinum neurotoxins (BoNTs) are the most poisonous substances known, with a human LD50 values estimated to be 1-3 ng/kg. BoNTs act with high specificity and potency to prevent neurotransmitter release in the presynaptic compartment, causing sustained paralysis and death by asphyxiation. The same characteristics that make BoNTs such effective therapeutic tools (persistence, exceedingly high potency, ease of use and ease of production) also puts them at high risk of misuse. Weaponized BoNT presents a significant civilian and force health protection concern and the neurotoxin is one of six agents (and the only toxin) to be labeled a Tier 1 threat agent. There is no post-exposure therapy to reverse BoNT intoxication. We were the first to demonstrate the use of stem cell-derived neurons as a functional model for BoNT research, and have since shown that ESNs, human stem cell-derived neurons, and primary mouse and rat CNS neurons recapitulate the unique toxin behaviors observed in vivo in motor neurons (McNutt, et al, 2011; Hubbard et al, 2012; Hubbard et al, 2015; Beske et al, 2016). Notably, intoxication of primary or derived neuron cultures eliminates spontaneous and evoked synaptic activity at femtomolar toxin concentrations, thereby producing a functional model of intoxication that allows for assessment of candidate therapeutics. We are currently using this model to (1) characterize mechanisms of intoxication and recovery from intoxication and (2) develop next-generation screening platforms based on the measured inhibition of synaptic activity to rapidly identify effective countermeasures. Based on these studies, we have identified several categories of chemical and biological entities that appear to transiently or permanently restore synaptic activity. We are currently validating these compounds ex vivo in phrenic nerve:diaphragm preparations using isometric contraction studies, as well as in single endplate electrophysiological recordings. Successful candidates are currently being translated to in vivo botulism models using novel assays based on behavioral and neurophysiological readouts. This work is supported by the Defense Threat Reduction Agency and two NIH R01s.
(2) Understand and mitigate nerve agent-induced CNS injury. Exposure to high doses of traditional nerve agents (such as soman, sarin or VX) causes an acute seizurogenic behavior in the CNS that results in significant neuropathology and in the long-term, elicits neuropsychiatric deficits. Neither the mechanisms responsible for seizure onset nor long-term neuropathologies are fully understood. Our lab utilizes a variety of techniques such as slice electrophysiology, cortical electroencephalograms, survival studies and immunohistochemistry to (1) understand acute synaptic changes associated with seizure onset, (2) identify therapeutically accessible systems that can mitigate seizure severity or otherwise decrease mortality, (3) identify novel therapeutic mechanisms that contribute to neurobehavioral deficits, and (4) test the effects of direct conversion of reactive glia into neurons on long-term histological and functional outcomes. While these studies have recently been started, we have some very exciting data implicating a previously unexplored CNS signaling system that has a strong protective effect against nerve agent injury. In animal sarin studies, agonizing this signaling pathway dramatically reduces mortality and post-exposure behavioral deficits, suggesting that it represents a novel therapeutic target. In additional studies we are using cortical EEGs to monitor the effects of first-line epilepsy drugs on long-term recurrent seizure activity in nerve agent exposed mice, with the goal of repurposing existing drugs as post-seizure treatments to reduce chronic morbidities. Finally, we are exploring the impact of nerve agent-induced status epilepticus on the fate and survival of neural stem cells. Collectively, this work is supported by a Chemical and Biological Defense fellowship and an NIH inter-agency agreement.
(3) Development of next-generation oxime countermeasures for nerve agent poisoning. Medical countermeasures for nerve agent exposure include an oxime, which disrupts interaction between nerve agents and acetylcholinesterase and allows the resumption of normal cholinergic neurotransmission. Lead candidate oximes suffer from several PK/PD concerns, including a poorly understood toxicity that limits their therapeutic index, poor systemic distribution, and selectivity for a subset of nerve agents. Thus there is a critical need for a safe, broad-spectrum oxime that is rapidly effective in both peripheral and central synapses. In collaboration with a medicinal chemist we are exploring the toxicities and potencies of oximes and oxime-like molecules to establish structure:function relationships, understand toxicophoric mechanisms and improve PK/PD characteristics, with the goal of developing the next-generation oxime countermeasure.
Primary neuron culture; Embryonic stem cell-derived neurons; Nerve agent; Botulinum neurotoxin; Drug discovery; Cell culture; Adeno-associated virus; Ex vivo nerve:muscle models; In vivo models; Neurophysiology; Whole-cell patch-clamp electrophysiology; Slice electrophysiology; Sulfur mustard; Planar electrode arrays; Cortical electroencephalograms; Seizure models; Anti-seizure drugs; Botulinum neurotoxin antidotes; Medical countermeasures; Nerve agents; Metabolic monitoring; Running wheel activity;