Fig 2: Effect of CA-AMPK expression on synaptic integrity, density, and autophagic markers.Primary neurons were infected at 17 DIV with a constitutively active form of AMPK (CA-AMPK) for 7 days. b Cytotoxicity was assessed by measuring the lactate dehydrogenase (LDH) released in cell media. Treatment with 0.9% Triton X-100 was used as a positive control. Results represent mean ± SD, n = 4. Cells lysates were analyzed by WB (a) and the ratio pACC/ACC was quantified (c). d–i Quantification of the pre-synaptic markers SNAP25 (d), Munc (e), and synapsin Ia (f) and the post-synaptic markers PSD-95 (g), GluN1 (h), and GluA1 (i). Results represent mean ± SD, n = 8. j Immunostaining of synaptophysin (SYP, red) and PSD-95 (green) in primary neurons infected at 17 DIV with the CA-AMPK for 7 days. k Quantification of pre-synaptic (red), post-synaptic (green), and synaptic (yellow) puncta upon CA-AMPK expression. Results represent mean ± SEM, n = 38 neurons counted from five independent experiments. Scale bar = 50 µm. l–p Analysis of the expression of proteins involved in autophagy pathway. Quantification of the ratios pRaptor/Raptor (m) and pULK/ULK (n) and of the expression level of p62 (o) and LC3 II proteins (p). Results represent mean ± SD, n = 6. Student’s t-test was performed for c–i and m–p experiments. One-way ANOVA with Bonferroni’s post hoc test was used for b, k experiments; *p < 0.05, **p < 0.01, ***p < 0.001 compared to Ctrl

Fig 3: Downregulation of excitatory receptor and postsynaptic transmission after BPA exposure. A,B) Representative immunoblot and corresponding densitometric analysis showing the ratios of expression amounts of NMDA receptor subtypes (NR1, NR2A, and NR2B to GAPDH (#p > 0.05, *p < 0.05, and #p > 0.05, respectively) and AMPA receptor subtypes (GluR1 and GluR2) to GAPDH in control and juvenile BPA treated groups (n = 8 rats per group, *p < 0.05 and p > 0.05, respectively); C,D) Excitatory synaptic transmission in BPA‐treated brain slices (10 × 10−6 m) was assessed by recording NMDA and AMPA receptor mediated current in the hippocampal CA1 areas, as shown by decreased EPSCNMDA, not EPSCAMPA, after acute BPA treatment (n = 8 rats per group, *p < 0.05 and p > 0.05, respectively).

Fig 4: Male rats express more GluA1-containing AMPA receptors and NMDA receptors on the postsynaptic membrane surface of TA-CA1 synapses.(A) Upper: representative Western blots of SLM tissue wedges sub-dissected from acutely prepared hippocampal slices show that male animals expressed more surface, but not total, GluA1 at TA-CA1 synapses. Lower: data summary of 3 independent membrane surface protein biotinylation experiments from 6 pairs of littermate male and female rats (male surface GluA1: 111.6 ± 3.5% of female, n = 6, t = 2.34, p < 0.05; male total GluA1: 93.5 ± 7.7% of female, n = 6, t = 0.85, p > 0.05). (B) Upper: representative Western blots of SLM tissue wedges sub-dissected from acutely prepared hippocampal slices show that male animals expressed more surface and total GluN1 at TA-CA1 synapses. Lower: data summary of 3 independent membrane protein biotinylation experiments from 6 pairs of littermate male and female rats (male surface GluN1: 121.2 ± 8.4% of female, n = 6, t = 2.47, p < 0.05; male total GluN1: 117.8 ± 7.3% of female, n = 6, t = 2.39, p < 0.05). *p < 0.05.

Fig 5: TSPAN5 regulates exocytosis of newly synthesised α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), possibly by preventing their degradation via the lysosomal pathway.(A) Schematic of the ARIAD-GluA2 construct. (B) In basal conditions (Berditchevski, 2001), ARIAD-GluA2 is retained in the endoplasmic reticulum (ER) due to the self-assembly properties of the conditional aggregation domain (CAD). Upon application (Charrin et al., 2002), the ARIAD ligand binds to CAD, inhibits self-assembly and allows the ARIAD-GluA2 to move to the Golgi where the endogenous Furin protease cleaves the CAD. ARIAD-GluA2 can now be loaded onto secretory vesicles (Charrin et al., 2014), transported along the dendrites, and subsequently exocytosed (Hemler, 2005). Application of an anti-myc antibody in the culture medium allows for the detection of the plasma membrane pool of GluA2 that was released from the ER after application of the ARIAD ligand. (C) Left panel: Confocal images of DIV20 rat cultured hippocampal neurons transfected at DIV12 with the ARIAD-myc-GluA2 construct and with a plasmid coding for GFP (green) and either scrambled, Sh-TSPAN5, or rescue, and immunostained with an anti-myc antibody in live staining conditions (magenta) 90 min after the application of the ARIAD ligand. Scale bar = 5 µm. Right panel: Quantification of the surface anti-myc mean intensity normalised to scrambled (scrambled 100±7.14; Sh-TSPAN5 71.44±6.81; rescue 101.4±7.92). n = 27–31 neurons per condition. One Way ANOVA, Newman-Kulspost hoc multiple comparison test.(D) Confocal images of secondary dendrites from DIV20 rat cultured hippocampal neurons transfected at DIV12 with either scrambled or Sh-TSPAN5 constructs, both co-expressing GFP. Neurons were treated for 90 min with either vehicle (H2O) or leupeptin (100 μM), fixed and immunostained for GLUA2/3 (magenta). Scale bar = 5 μm. (E) Relative quantification of GluA2/3 staining mean intensity (GluA2/3 mean intensity: scrambled vehicle 31.25±2.43; scrambled leupeptin: 36.95±2.25: Sh-TSPAN5 vehicle: 24.51±1.35; Sh-TSPAN5 leupeptin 33.3±2.22). n = scrambled vehicle, 20; scrambled leupeptin, 20; Sh-TSPAN5 vehicle, 20; Sh-TSPAN5 leupeptin, 20 neurons. One Way ANOVA, Newman-Kulspost hoc multiple comparison test. (F) Left panel: Confocal images of DIV20 rat cultured hippocampal neurons transfected at DIV12 with the ARIAD-myc-GluA1 construct and with a plasmid coding for GFP (green) and either scrambled, Sh-TSPAN5, or rescue, and immunostained with an anti-myc antibody in live staining conditions (magenta) 90 min after the application of the ARIAD ligand. Scale bar = 5 µm. Right panel: Quantification of the surface anti-myc mean intensity normalised to scrambled (scrambled 95.32±5.07; Sh-TSPAN5 52.49±4.95; rescue 81.064±6.2). n = 27–29 neurons per condition. One Way ANOVA, Newman-Kulspost hoc multiple comparison test.Values represent the mean ± SEM. *=p < 0.05, **=p < 0.01, ***=p < 0.001. Figure 7—source data 1.Individual data values for the graphs presented in panels C, E, and F.