bioRxiv preprint doi: https://doi.org/10.1101/237370; this version posted December 28, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Running Title: Broad Spectrum Proteomics Analysis of the Inferior Colliculus following Acute 2 Hydrogen Sulfide Exposure 3 Broad Spectrum Proteomics Analysis of the Inferior 4 Colliculus following Acute Hydrogen Sulfide Exposure 5 Dong-Suk Kim1, Poojya Anantharam1a, Andrea Hoffmann2, Mitchell L. Meade3, Nadja Grobe3, 6 Jeffery M. Gearhart2, Elizabeth M. Whitley4, Belinda Mahama1b, Wilson K. Rumbeiha1* 7 1Veterinary Diagnostic & Production Animal Medicine, Iowa State University, Ames, IA, US 8 2Henry M Jackson Foundation on contract 711HPW/USAFSAM/FHOF, Wright Patterson Air 9 Force Base, Dayton, OH, US 10 3711HPW/RHDJ, Wright Patterson Air Force Base, Dayton, OH, US 11 4Pathogenesis, LLC, Gainesville, FL, US 12 *Corresponding Author’s Contact Information:[email protected], 515-294-0630 13 Key Words: Hydrogen Sulfide, Brain Injury, Proteomic Profiling, Proteomic Analysis, TMT 14 labeled LC-MS/MS, Neurodegeneration 15 Present addresses: 16 aPoojya Anantharam: Medical Countermeasures, MRI Global, Kansas City, MO, US 17 bBelinda Mahama: Neuroscience, Brown University, Rhode Island, Providence, US Cleared, 88PA, Case # 2017-5041, 18 Oct 2017. 1 bioRxiv preprint doi: https://doi.org/10.1101/237370; this version posted December 28, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 18 Abstract: Acute exposure to high concentrations of H2S causes severe brain injury and long-term 19 neurological disorders. The mechanisms of H2S-induced neurodegeneration are not known. To 20 better understand the cellular and molecular mechanisms of H2S-induced neurodegeneration we 21 used a broad-spectrum proteomic analysis approach to search for key molecules in H2S-induced 22 neurotoxicity. Mice were subjected to acute whole body exposure of up to750 ppm of H2S. The 23 H2S-treated group showed behavioral motor deficits and developed severe lesions in the inferior 24 colliculus (IC), part of the brainstem. The IC was microdissected for proteomic analysis. Tandem 25 mass tags (TMT) liquid chromatography mass spectrometry (LC-MS/MS)-based quantitative 26 proteomics was applied for protein identification and quantitation. LC-MS/MS was able to 27 identify 598, 562, and 546 altered proteomic changes for day 1 (2 h post H2S exposure), day 2, 28 and day 4 of H2S exposure, respectively. Mass spectrometry data were analyzed by Perseus 29 1.5.5.3 statistical analysis, and gene ontology heat map clustering. Quantitative real-time PCR 30 was used to confirm some of the H2S-dependent proteomics changes. Taken together, acute 31 exposure to H2S induced behavioral motor deficits along with progressive neurodegeneration 32 including disruption of several biological processes in the IC such as cellular morphology, 33 energy metabolism, and calcium signaling. The obtained broad-spectrum proteomics data may 34 provide important clues to elucidate mechanisms of H2S-induced neurotoxicity. 35 Cleared, 88PA, Case # 2017-5041, 18 Oct 2017. 2 bioRxiv preprint doi: https://doi.org/10.1101/237370; this version posted December 28, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 36 Highlights: 37 Mice exposed to H2S recapitulated H2S-induced neurotoxicity manifested in humans. 38 The IC in the mouse brain is the most sensitive to H2S-induced neurodegeneration. 39 Proteomic expressions of key proteins were validated at transcription level. 40 Several biological pathways were dysregulated by H2S exposure. 41 42 Cleared, 88PA, Case # 2017-5041, 18 Oct 2017. 3 bioRxiv preprint doi: https://doi.org/10.1101/237370; this version posted December 28, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 43 1. Introduction 44 Hydrogen sulfide (H2S) is a highly neurotoxic colorless gas with a “rotten egg” odor (Chou et 45 al., 2016). It is as an environmental pollutant that causes occupational hazards in a variety of 46 industrial processes including the oil and gas industry, intensive animal farming operations, 47 sewer and waste water treatment plants, pulp and paper plants, and gas storage facilities, among 48 several others (Chou et al., 2016). It has been estimated that there are more than 1,000 reports of 49 human exposures to H2S each year in the United States (Chou et al., 2016). Besides accidental 50 H2S poisoning in industrial settings, intentional acute exposure to high concentrations of H2S as 51 a means of suicide has been increasingly observed in Western and Asian societies (Morii et al., 52 2010; Reedy et al., 2011). The raw chemical ingredients used to generate H2S in such 53 circumstances are readily accessible in local stores (Morii et al., 2010). More significantly, H2S 54 is listed as a high priority chemical by the US Department of Homeland Security because of its 55 potential to be misused in nefarious acts such as chemical terrorism, particularly in confined 56 spaces such as the underground transit systems. Although H2S is toxic at high concentrations, it 57 is also beneficial at physiologic concentrations. For this reason, there is also tremendous interest 58 in potential therapeutic applications of H2S for treatment of a number of human disease 59 conditions. Proposed pharmacological uses of H2S include ulcer treatment, ischemic reperfusion 60 injury, as an anti-inflammatory for treatment of Crohn’s disease, endotoxin induced 61 inflammation, arterial hypertrophy, visceral pain, Parkinson’s disease and cancer among others 62 (Szabo, 2007; Popov, 2013). Furthermore, H2S was shown to activate nitric oxide (NO) synthesis 63 through induction of endothelial nitric oxide synthase. Similar to NO, H2S serves as a 64 gasotransmitter and signaling molecule in the CNS and regulating vasodilation (Tan et al., 2010). Cleared, 88PA, Case # 2017-5041, 18 Oct 2017. 4 bioRxiv preprint doi: https://doi.org/10.1101/237370; this version posted December 28, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 65 Like most toxicants, the toxicity of H2S exposure is dose-dependent, but H2S characteristically 66 has a steep-dose response curve (Guidotti, 1996). At concentrations below 30 parts per million 67 (ppm), H2S causes headache, coughing and throat irritation. At 150 ppm H2S induces olfactory 68 fatigue and temporary loss of smell after 5-60 min of exposure (Wasch et al., 1989). 69 Concentrations higher than 500 ppm cause headache, dizziness, unconsciousness, and respiratory 70 failure. At high concentration above 1,000 ppm H2S can lead to immediate loss of consciousness, 71 commonly called “knock-down”, and ultimately seizures, and sudden death (Chou et al., 2016). 72 Typically, acute exposure to high concentrations of H2S is associated with high mortality withing 73 a few hours post-exposure (McCabe and Clayton, 1952). Timely rescue of victims can prevent 74 disaster and allow victims to fully recover. Currently, there is no effective antidote to treat 75 victims of H2S gas exposure and the mortality rate remains high (Lindenmann et al.; Herbert, 76 1989; Vicas, 1989; Smith, 1997; Lindenmann et al., 2010). In addition, delayed neurological 77 disorders, which can last for many years, are commonly reported in survivors of acute H2S 78 exposures (Matsuo et al., 1979; Parra et al., 1991; Tvedt et al., 1991a; Tvedt et al., 1991b; 79 Snyder et al., 1995; Kilburn, 2003; Woodall et al., 2005). These delayed neurological sequelae 80 are incapacitating, and require prolonged medical attention that lacks defined medical 81 interventions. 82 The development of effective therapeutics requires a good understanding of the molecular 83 mechanisms and pathways of H2S-induced neurodegeneration and neurological sequelae. These 84 mechanisms remain largely unknown. There is an acute need for countermeasures for treatment 85 of mass civilian casualties of acute H2S poisoning in the field, such as following catastrophic 86 industrial meltdowns or intentional terrorist activities. Elucidating molecular mechanisms Cleared, 88PA, Case # 2017-5041, 18 Oct 2017. 5 bioRxiv preprint doi: https://doi.org/10.1101/237370; this version posted December 28, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 87 underlying H2S-induced neurotoxicity is essential in developing suitable targeted therapeutics to 88 counter both acute and delayed neurotoxic effects of H2S poisoning in humans. 89 We recently developed a relevant inhalational animal model that exhibits the clinical, 90 pathological, and motor behavioral symptoms of H2S exposed survivors (Anantharam et al., 91 2017). The objective of this study was to build on our previous work and investigate proteomic 92 changes and altered gene expression in a mouse model of acute H2S-exposure to identify novel 93 toxic mechanisms. In prior studies, we discovered that the central inferior colliculus (IC) region 94 of the brainstem is the most sensitive brain region to H2S-induced neurodegeneration 95 (Anantharam et al., 2017).