Experimental procedures

Mouse proteomics assays are described in detail in Marques et al.(2022d). Briefly, a group of healthy male C57BL/6J mice was treated with 1 mg kg-1 MTK by oral gavage, once a day, for a week. Brain, hippocampus, and prefrontal cortex were collected and used for protein extraction. Samples were analysed by ultra-performance liquid chromatography coupled to high resolution mass spectrometry with electrospray ionization UPLC‑HRMS-ESI and the raw data were deposited in public data repositories (Marques et al. , 2022b). The well-characterized C57BL/6J mouse strain is the strain more commonly used for safety and toxicity assessment, and is also refractory to the development of many tumours, making it an ideal strain for metabolic and omics studies on non-tumorigenic toxic effects (Kalueff and Nguyen, 2014; Mouse Genome Sequencing Consortium, 2002; Sarsani et al. , 2019).
A detailed description of neuron proteomic assays is also available in Marques et al. (2022d). A chicken neuron-enriched cell model was exposed to MTK and cells were collected for protein extraction. Samples were analysed by UPLC‑HRMS-ESI and raw data deposited in public data repositories (Marques et al. , 2022a). Brain-expressed chicken amyloid-β has a higher homology to the human form than that of mouse. Since amyloid-β is one the proteins involved in Alzheimer’s disease, for which the repurposing of MTK is being considered, we developed an optimised chicken-based neuron model (Carrodeguas et al. , 2005) for assessment of MTK‑driven proteomics alterations.

Data processing

Raw data were re-processed according to the parameters presented in Marques et al. (2022d). Mouse and chicken proteomics data were analysed on MaxQuant software v2.0.3.0 (Cox and Mann, 2008; Tyanovaet al. , 2016a) using the internal search engine Andromeda (Coxet al. , 2011), and Uniprot (UniProt Consortium, 2020) databases restricted to specific groups of proteins from Mus musculus(Proteome UP000000589; keywords “ADRs (anxiety + stress + sleep + depression + suicide) + apoptosis + inflammation + leukotrienes + metabolism + microglia + neurons + repurposing (amyloid, synuclein, tau, APP, BACE, Psen1, Psen2, nicastrin, neprilysin, adam 17)” + tissue name ”) or Gallus gallus (Proteome UP000000539, keywords “ADRs (anxiety + stress + sleep) + inflammation + leukotrienes + neurons + repurposing (amyloid, synuclein, tau, APP, BACE, Psen1, Psen2, nicastrin, neprilysin, adam 17 )”). MaxQuant output was processed using Perseus v1.6.15.0, with default settings (Tyanova et al. , 2016b). Significantly altered proteins (|fold change| ≥ 1.5 and p  < 0.05) were annotated using PANTHER v16.0 andMus musculus or Gallus gallus reference lists (Mi et al. , 2021).