Fig 2: DIO alters hypothalamic VLDLR and ApoER2 mRNA and RELN protein levels.A, Relative hypothalamic expression of RELN in control (CTRL; n = 7) and DIO (n = 8) mice. B, Relative hypothalamic expression of ApoER2 in CTRL (n = 7) and DIO (n = 8) mice. C, Relative hypothalamic expression of VLDLR in CTRL (n = 7) and DIO (n = 8) mice. Individual data points from qPCR analyses are shown for each mouse. D, Western blot of FL-RELN, NT-RELN protein fragments, ApoER2, and β-actin in CTRL (n = 6) and DIO (n = 6) mice E, Relative optical density of Nt-RELN (F), FL-RELN and (G) ApoER2 protein levels in CTRL and DIO mice normalized to β-actin. (Error bars indicate ±SEM; student's t-test *p < 0.05).

Fig 3: CF-RELN alters pre- and post-synaptic inputs onto ARH-POMC-EGFP neurons. A, Representative traces from a voltage-clamp experiment showing both reduced and enhanced mIPSC frequency after bath application of CF-RELN (100 nM) in POMC-EGFP neurons. B, Normalized mIPSC frequency (n = 14) before and after treatment of CF-RELN (0.9 ± 0.1-fold; Bold line, cumulative mean change; one-way ANOVA with pairwise analysis) C, Fold change of mIPSC frequency in CF-RELN reduced (0.8 ± 0.1-fold; left) and enhanced (1.1 ± 0.04-fold; right) as determined by KS-test. D, Baseline mIPSC frequency of neurons which CF-RELN reduced (R; n = 5) or enhanced (E; n = 9) mIPSC frequency E, Mean amplitude of mIPSCs before and after CF-RELN application (n = 14). Between-cell analysis performed using a paired t-test or mixed-effects model with Tukey's post-hoc analysis. (Error bars indicate ± SEM *p < 0.05).

Fig 4: DIO alters actions of CF-RELN on mIPSC amplitude in ARH-POMC-EGFP neurons, but not membrane potential. A, Representative trace from a voltage-clamp experiment showing reduced mIPSC frequency after bath application of RELN (100 nM) in a POMC-EGFP neuron from DIO mice. B, Basal mIPSC frequency in POMC-EGFP neurons from chow fed (n = 14) and DIO mice (n = 15) C, Fold change of mIPSC frequency in neurons which CF-RELN reduced (R; 0.8 ± 0.0-fold; n = 7) or enhanced (E; 1.1 ± 0.1-fold; n = 8) mIPSC frequency as determined by KS-test D, Mean amplitude of mIPSCs in POMC-EGFP neurons from chow fed (n = 14) and DIO mice (n = 15) before and after RELN application. E, Representative current-clamp trace of showing membrane depolarization of a POMC-EGFP neuron in a DIO mouse after bath application of CF-RELN (100 nM). (F) Mean and change in (G) membrane potential before and after application of CF-RELN in DIO mice (n = 10). (Between-cell analysis using a Mixed-effects model with Tukey's post-hoc analysis *p < 0.05).

Fig 5: Recombinant CF-RELN stability and in vitro ApoER2 binding affinity. A, Schematic of the full-length RELN protein (N: N terminus, SP: signal peptide, F-SL: F-spondin like domain, U: Unique domain, A/B: subdomains of the eight RELN repeats, C+: positively charged C-terminus) illustrating the three fragments and ApoER2/VLDLR binding site. B, Coomassie stain of CF-RELN without (NR) and with (R) the presence of reducing agent (M: molecular weight marker). C, Absorption as measured by optical density (OD) for binding of in-house and R&D Systems CF-RELN to biotinylated ApoER2 D, ApoER2 competitive binding assay for CF-RELN (R&D Systems) expressed as molar ratio of non-biotinylated (cold) ApoER2 to biotinylated ApoER2. (Error bars expressed as ± SEM * p < 0.05).