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Proteomic analysis of brain regions of adult Long-Evans rats exposed to kainic acid
Citation:
Pitzer, E., G. Jung, W. Padgett, W. Winnik, C. Ganta, N. Clayton, H. Jensen, R. Herbert, AND D. Herr. Proteomic analysis of brain regions of adult Long-Evans rats exposed to kainic acid. Society of Toxicology, San Diego, California, March 27 - 31, 2022.
Impact/Purpose:
The impact of this study is that it is the first use of proteomics to assess excitatory neurotoxicity using Kainic acid exposure. It will allow for in vitro to in vivo extrapolation. This study supports the CSS 20.4 research goals.
Description:
We are using proteomics to measure changes in protein signatures produced by prototypical neurotoxicants, allowing better understanding of the molecular events produced by these compounds. Adult male Long Evans rats were treated with kainic acid (KA; 6 mg/mL, s.c., chosen not to produce myoclonic seizures), vehicle (VC; buffered saline, 0 mg/mL), or were non-injected (cage controls, CC). At 3 or 24h post-exposure, the rats were perfused with DPBS and brain regions collected and stored at -80°C. Hippocampal samples were assessed for proteomic content using Orbitrap LC-MS, and proteins were identified and processed using Proteome Discover. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) software (differential protein cutoff: ≤ log2(0.8), ≥ log2(1.2), FDR p-value ≤ 0.05). A total of 1894 and 1770 proteins were identified for mapping with IPA, at 3 and 24 h respectively. Of those proteins, 199 and 103 were altered by KA treatment at 3 and 24h, respectively. At 24h the top proteomic inhibited pathways included synaptogenesis signaling pathway, G Beta Gamma signaling, and Ephrin receptor signaling. Activated pathways at 24h included PTEN signaling, semaphoring neuronal repulsive signaling pathway, and autophagy. Proteins impacted in these pathways included, RAC (Rho family)-alpha serine/threonine-protein kinase (AKT; downregulated, in all above pathways), Ras GTPase (RAS; downregulated, in all above pathways except autophagy), and Growth factor receptor-bound protein 2 (GRB2; upregulated, pathways: synaptogenesis signaling, G Beta Gamma, ephrin receptor, PTEN), as well as additional up- or downregulated proteins. An additional set of rats were treated and sacrificed at 3, 24, or 48h following KA exposure (0 or 6 mg/mL), perfused with DPBS then 4% paraformaldehyde (PFA) and fixed in situ with 4% PFA. Hematoxylin and Eosin-stained hippocampal sections revealed no significant microscopic lesions. The proteomic signature revealed several pathways, including neuronal signaling and cell death to be impacted, in the absence of overt histological changes. This is an abstract of a proposed presentation and does not necessarily reflect US EPA policy.