Rhbdnf

Induced pluripotent stem cells were differentiated to neurons using a protocol that was previously published (47 (link)). Neurons were quickly thawed at 37°C and seeded at a density of 200,000 cells/cm² in neuronal differentiation media [consisting of 1:1 Advanced DMEM/F12 media (with GlutaMAX I) (Thermo Fisher Scientific) and Neurobasal media (Thermo Fisher Scientific) + 1% B27 supplement without vitamin A (Thermo Fisher Scientific), 1% N2 supplement (Thermo Fisher Scientific), 50 µg/ml Beta-Mercaptoethanol (Thermo Fisher Scientific), 10 U/ml Penicillin-Streptomycin (Thermo Fisher Scientific), 20 ng/ml rhBDNF (PeproTech), 10 ng/ml rhGDNF (PeproTech), 100 µM Aa2-P (Sigma Aldrich), 500 µM cAMP (BIOLOG Life Science), and 1 µg/ml murine laminin (Roche), supplemented with 10 µM ROCKi (Y27632, StemCell Technologies) for seeding]. After a 100% media change one day after seeding, 50% of the media was changed twice a week. After 14 days, macrophage precursor cells were added at a density of 160,000 cells/cm². Therefore, the media was replaced with a macrophage precursor cell suspension in N2 media [Advanced DMEM/F12 + 1% N2 supplement, 10 U/ml Penicillin-Streptomycin, 2 mM Glutamax, 50 µg/ml β-ME, 100 ng/ml rhIL34 (Miltenyi Biotech), and 10 ng/ml rhGM-CSF (biotechne)]. Half of the media was changed twice a week for two additional weeks.

Parental SH-SY5Y cells and transfected cells were analyzed both under basal conditions and following neuronal differentiation in all-trans Retinoic Acid-Neurobasal medium (RA-NBM) medium (Pezzini et al., 2017 (link)). In brief, cells were pre-differentiated in DMEM/HIGH supplemented with 5% FBS and 10 μM RA (Sigma-Aldrich) for 6 days, and then grown for further 3 days in Neurobasal medium (GIBCO, LT) enriched with 50 ng/ml recombinant human BDNF (rhBDNF, Peprotech), 2 mM Dibutyryl-cyclic AMP (db-cAMP; Sigma-Aldrich), 20 mM KCl, B27 supplement and 1% GlutaMAX (GIBCO, LT). Control (not treated, NT) cells were cultured in parallel under basal conditions at the same FBS concentration (5%). Cells were seeded in T₇₅ flasks or on coverslips coated with ECMax gel (Sigma-Aldrich) at the same density of 8 × 10³ cells per cm², grown for 24–48 h and then exposed to RA-NBM treatment. Media were routinely changed every 2–3 days; the morphological features of control and CLN1 transfected cell lines were monitored under an Axio Vert.A1 inverted microscope (Zeiss). The images were acquired by True Chrome HD II cam system. Cells grown under basal conditions were harvested after 6 days to avoid the culture over-growth.

Anandamide (AEA, #1017; Tocris, Bethesda, MD, USA), arachidonyl-2′-chloroethylamide (ACEA, #1319, Tocris, USA), WIN 55,212-2 mesylate (WIN, #1038; Tocris), capsaicin (CPS, #0462; Tocris, USA), AM251 (#1117; Tocris), TRKB.Fc (#688-TK-100; R&D Systems, Minneapolis, MN, USA), capsazepine (CZP, #0464; Tocris), k252a (#05288, Sigma-Aldrich, USA) and recombinant-human brain-derived neurotrophic factor (rhBDNF, #450-02; Peprotech, Rocky Hill, NJ, USA) were used. AEA and ACEA were dissolved in Tocrisolve (#1684; Tocris). WIN and AM251 were suspended in 2%Tween 80, and k252a was diluted in DMSO 0.2%, and dissolved in sterile saline for ip injections. CPS, CZP or AM251 were diluted in DMSO (at 1,000×the final concentration used in the assays) for in vitro experiments. BDNF and TRKB.Fc were dissolved in phosphate buffered saline (PBS). The doses of the drugs used to intracerebral infusions were based on previous works which have shown those doses being effective to change the animal behavior (Casarotto et al., 2010 (link); Casarotto et al., 2012 (link); Casarotto, de Bortoli & Zangrossi Jr, 2012 (link); Fogaça et al., 2012 (link); Terzian et al., 2014 (link)).