2-Materials and methods

2.1 Animal model C57Bl/6 Naglu−/− (MPSIIIB) mice were purchased from Jax Lab (stain ref #003827). This model was initially obtained by introducing a neomycin resistance cassette into the exon 6 of Naglu gene, interrupting therefore the production of full length active NAGLU enzyme (Li et al., 1999). The experiments were conducted in male and female wildtype (WT) and MPSIIIB mice, ensuring that we included an equal number of males and females in each experiment whenever possible. They were fed standard laboratory chow for 2 or 9 months, with food and water provided ad libitum. All experiments were performed in accordance with European Committee Standards concerning the care and use of laboratory animals and were approved by the local ethical committee (n°122) and by the French Ministry of Higher Education and Research (n° 2020273122248406V4). 2.2 Mouse brain tissue processing The mice were first subjected to euthanasia by injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Blood samples were then collected by cardiac puncture, and brains were removed after heart perfusion with PBS. Isolated brains, were either harvested and frozen at −80°C for further analysis, or immediately processed for immunohistochemistry processing. 2.3 Brain sections immunohistochemistry Brain samples were embedded in paraffin and sectioned into 5 µm slices. The brain sections were then deparaffinized and rehydrated using histoclearII® baths and antigen retrieval was performed using microwave heated 1x citrate buffer. The sections were then incubated with blocking solution (HRP008DAB, Zytomed) and with peroxide block solution (E41-100, Diagomics) to avoid non-specific binding of antibodies or of 3,3-diaminobenzidine (DAB) staining. Primary antibodies were used at 1:400 dilutions for anti-FPN (P1-21502, Novus Biological) and anti-FTH (DF6278, Affinity Biotech). The immunoreactive staining were revealed with the streptadivin-HRP conjugated secondary antibodies and DAB solution. 2.4 Tissue iron quantification 100 mg of these brain tissues were cut into 4 to 6 pieces using a non-metallic scalpel. They were placed in a microtube washed with 0.5 M HCl. After adding 400 µL of protein precipitation solution (H20, trichloroacetic acid, HCl C°36%–38%, Thioglycolic acid), the samples were placed in a double boiler at 65°C for at least 24 h. Once the liquid supernatant had been recovered and weighed, the equilibration solution (sodium acetate pH4.5) diluted 1:10 was added. The concentration of ferric iron, including free and bound iron, was then measured using an automated system (Kit Iron #SI8330, Ferrozine, Rendox Daytona+, Randox Laboratories, France). 2.5 RNA extraction and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) Total RNA was isolated using the SV Total RNA Isolation System Kit (Z3103, Promega) according to the supplier’s recommendations. The reverse transcription reaction was performed using 1.5 µg of total RNA in a final volume of 20 μL. qPCR was performed in duplicate using the SsoFast EvaGreeen PCR-mix (1725204, BioRad) and specific primers for TLR4, ZIP14, iNOS, nNOS, SIRT3, SOD2, ACSL4, LPCAT3 and ARPO transcripts (Table 1). Results were normalised to transcripts of the brain specific ARPO housekeeping gene. Results were expressed in arbitrary units as a fold change relative to the control sample using a 2−ΔΔCT calculation (ΔΔCt = ΔCt exposed − mean ΔCt control). qPCR and statistical analyses were performed using StepOne Software v2.3 and PRISM (Prism 10 Version 10.1.1).