DOI: 10.25345/c59c6sb49
title: MassIVE MSV000093047 - Chlorpyrifos induces autophagy in murine GnRH neurons by repressing the mTOR pathway and affecting androgen and estrogen alpha/beta receptor expression
title: Chlorpyrifos (CPF) is a widely used pesticide inducing neurodevelopmental and reproductive adverse effects. Little is known about the underlying mechanisms, especially in hypothalamus. We investigated CPF mode of action at human relevant concentrations (1 nM_100 nM) in immortalized murine hypothalamic GnRH neurons (GT1_7), an elective model to study central derangement of the hypothalamus_pituitary_gonads (HPG) axis. Treated cells were examined for cell vitality, proliferation, spheroid morphology, apoptosis, neuron functionality, gene expression, Transmission Electron Microscopy, immunoreactivity and proteomics profiles. CPF dose_dependently decreased cell vitality. At 100 nM, CPF induced autophagy and mitochondria damage, and inhibited GnRH gene expression and secretion. Spheroid morphology was impaired and immunoreactivity of MAP2 neuron marker decreased with the dose. Estrogen Receptor alpha and beta (ERalpha, ERbeta), Androgen Receptor (AR), aromatase and oxytocin receptor gene expression was non_monotonically induced by CPF but with different patterns. The differentially expressed proteins enriched the Autophagy, mTOR signaling and Neutrophil extracellular trap (NET) formation pathways, supported by the dose_dependent decrease of mTOR immunoreactivity. A core module of interacting proteins featuring ERalpha_AR_mTOR and proteins of the NET pathway was identified. Overall, our results support CPF as inhibitor of the mTOR pathway, possibly involving ERalpha_AR signaling, leading to autophagy of GnRH neurons, thus raising concern on possible adverse effects on HPG axis. 30ug of proteins were subjected to a bottom_up proteomics workflow: in the fist step they were treated with TCEP and IAM, for the reduction and alkylation of the cysteines, respectively. Then, proteins were precipitated using a solution of methanol, acetone and ethanol (25, 25 and 50 v_v) at minus 20C over_night and after a centrifugation at maximum speed for 15 min at 4C the resulting pellet was resuspended in urea and ammonium bicarbonate and the proteins were digested adding trypsin, at a substrate to enzyme (S_E) ratio of 50 (w_w) for 16 h at 37 C on Thermo Mixer heat block. The day after, formic acid was added to block digestion and 20uL of the resulting peptide mixture was injected in an Ultimate 3000 UHPLC coupled with an Orbitrap Fusion Tribrid mass spectrometer. Peptides were desalted on a trap column and then separated on a 45cm long silica capillary, packed in house with a C18, 1,9um, 100 A resin. The analytical column was encased by a column oven (Sonation, 40C during data acquisition) and attached to a nanospray flex ion source. Peptides were separated on the analytical column by running a 180 min gradient of buffer A (95water, 5acetonitrile, and 0,1 formic acid) and buffer B (95acetonitrile, 5 water, and 0,1 formic acid), at a flow rate of 250 nl min. The mass spectrometer was operated in positive ion mode and precursor ion scanning was performed in the Orbitrap analyzer (FTMS; Fourier Transform Mass Spectrometry) in the scan range of m_z 350 1550 with 120K resolution. Data_dependent acquisition was performed in top_speed mode (3 s long maximum total cycle): the most intense precursors were selected through a monoisotopic precursor selection (MIPS) filter and with charge greater than1, quadrupole isolated and fragmented by higher energy collision dissociation (HCD) (30 collision energy). Product ion spectra were recorded in the ion trap (ITMS) with a rapid scan rate. Peptide spectra were searched in Proteome Discoverer 2,4 (Thermo Fisher Scientific) using Sequest HT as search engine against Mus Musculus database from UniProtKB_Swiss_Prot (Release 2022; 17090 sequences). Spectral matches were filtered using Percolator node, based on q values, with 0.01 false discovery rate (FDR), based on a target_decoy approach. Only master proteins were taken into account and only specific trypsin cleavages with two miscleavages were admitted. Cysteine carbamydomethylation was set as static modification, while methionine oxidation and N_acetylation on protein terminus were set as variable modifications. Precursor mass tolerance was set to 15 ppm while the MS_MS match tolerance was set 0.6 Da. Quantification was based on precursor intensity of unique and razor peptides using match between runs option.