Fig 2: Differences in lateral amygdala neuron morphology are associated with dysregulated gene transcription and protein expression. (a) Morphological analysis of reconstructed neurons from lateral amygdala of WT (n=26) and SPRED2 KO mice (n=22) revealed a higher total spine number on dendritic branches of branch orders 1–4. (b) Spine density per 10 µm of dendritic length was also higher within these branch orders. (c) Exemplary Western blot analyses showed changed expression levels of different pre- and postsynaptic proteins in the amygdala of SPRED2 KO mice in comparison to WT controls. Protein levels of PSD95, mGluR5, mGluR2, and ERC1 were increased, those of bassoon were decreased. Protein expression of Rab3A, Rab6, a-tubulin, ß-tubulin and GAPDH was unaltered; GAPDH was used as loading control. (d) Quantified signals of investigated synaptic proteins after normalization to GAPDH confirmed changed expression levels in the amygdala of SPRED2 KOs (n=11) compared with WTs (n=11). (e) Quantitative RT-PCRs with mRNA isolated from amygdala of SPRED2 KO mice (n=11) and WT controls (n=11) using gene-specific primers confirmed the increased expression of PSD95, mGluR5, mGluR2, and ERC1 on mRNA level. Data are mean±s.e.m; *P<0.05, **P<0.01, ***P<0.001. KO, knockout; n.s., not significant; WT, wildtype.

Fig 3: PRS effects on nigrostriatal dopaminergic transmission throughout the lifespan.Adult PRS rats showed fewer TH-expressing cells than age-matched controls, whereas aged rats in both groups showed fewer TH+ cells than did 4-month-old adult rats (Fig. 5A; n = 7–8 rats/group). 3H-DA release evoked by high concentrations of 12 K+ in striatal synaptosomes was significantly reduced by PRS in both age groups. Aging (21-month-old rats) reduced DA release in both unstressed and PRS rats (Fig. 5B; n = 5 rats/group). No changes were observed in the striatal levels of TH and A2AR (n = 4–8 rats/group), while an age effect was observed in D1R in both unstressed and PRS groups, with lower levels than those in adult rats (n = 4–8 rats/group). Aging reduced DAT protein expression only in unstressed rats (n = 4–8 rats/group) and increased D2R protein levels in both unstressed and PRS rats (n = 4–8 rats/group) (Fig. 5C). PRS reduced syntaxin-1 levels in both ages, but mainly in old rats. PRS also reduced Rab3a levels in both age groups (n = 5–8 rats/group) but mainly in adult rats. In both groups, aging led to higher synaptophysin (SYP) and SNAP-25 levels than those in adults (n = 5–8 rats/group). Neither aging nor PRS did not affected munc-18 protein expression (n = 5–8 rats/group) (Fig. 5D). CONT vs. PRS *p < 0.05; **p < 0.01; ***p < 0.001; adult vs. aged ###p < 0.001.

Fig 4: Atrial Natriuretic Peptide Secretion From Cardiomyocytes is Dependent on Rab3gap1 GAP Activity(A) Representative images of RNCMs transduced with adenoviruses for myc-tagged wildtype Rab3gap1 (Rab3gap1), a myc-tagged-GAP -deficient Rab3gap1 mutant (Rab3gap1R728A), or ß-galactosidase control (ßGal) and immunostained for myc (Rab3gap1, green) and GM130 (Golgi marker, red). (B) Representative images of RNCMs transduced with adenoviruses for GFP-Rab3a and either Rab3gap1, Rab3gap1R728A or ßGal and immunostained for GM130 (Golgi marker, red). (C) Pearson’s correlation coefficients for colocalization of Rab3gap1 and GM130 in the indicated groups of RNCMs. n = 3 independent experiments with 60 to 90 cells analyzed for each experiment. (D) Quantification of GFP-Rab3a relative fluorescence intensity. n = 3 independent experiments with 60 to 90 cells analyzed for each experiment. (E) Immunoblotting and quantification of (F) Rab3a-GTP and (G) total Rab3a levels in RNCMs transduced with adenoviruses expressing ßGal, Rab3gap1, or Rab3gap1R728A. Immunoblotting of (H) secreted and (I) intracellular ANP in RNCMs transduced with adenoviruses expressing ßGal, Rab3gap1, or Rab3gap1R728A. Coomassie staining or immunoblotting for Gapdh were used as loading controls in H and I, respectively. Quantification of (J) secreted and (K) intracellular ANP levels in the indicated groups of RNCMs as shown in H and I, respectively. n = 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, 1-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SD. Abbreviations as in Figures 1 and 2.

Fig 5: ATG9A vesicle localization in neurons.a–c Mouse hippocampal neuronal cultures were transfected as indicated and imaged at DIV14-17. a Representative confocal images (top), and corresponding line-scan analysis (bottom) of neuronal cultures expressing synaptophysin-HA with ATG9A-EGFP or mCherry-SCAMP5. Synaptophysin-HA was detected by anti-HA immunofluorescence. b Colocalization analysis calculated by Pearson’s coefficients. Values are means ± SEM. **p < 0.01 by the two-sided Student’s t-test (all boutons in 8 field of view (121 µm x 121 µm) for each condition from two independent cultures were quantified). c Localization of ATG9A-EGFP, synaptophysin-HA and mCherry-synapsin in presynaptic varicosities of an axon. Synaptophysin-HA was visualized by anti-HA immunofluorescence. d–g Mouse hippocampal neuronal cultures were fixed and stained as indicated at DIV14-17 to visualize endogenous localization of ATG9A in neurons. d, e Representative confocal images showing immunoreactivity for endogenous ATG9A, synaptophysin and VAMP2 (d) or Rab3A (e) in cultured hippocampal neurons. Corresponding line-scans are shown at the bottom. f, g Colocalization analysis. Values are means ± SD; N.S., not significant; **p < 0.01 by one-way ANOVA with the Tukey’s HSD post hoc test (12 images (121 µm x 121 µm for each) from two independent cultures were analyzed). Source data are provided as a Source Data file. Scale bars, a = 5 µm, c = 10 µm (1 µm for high-magnification images), d and e = 2 µm. p-values (b): 9 × 10?9, (f): 0 [(Syph vs. VAMP2) vs. (Syph vs. ATG9A)], 0 [(Syph vs. VAMP2) vs. (VAMP2 vs. ATG9A)], 0.8080 [(Syph vs. ATG9A) vs. (VAMP2 vs. ATG9A)]. (g): 0 [(Syph vs. Rab3A) vs. (Syph vs. ATG9A)], 0 [(Syph vs. Rab3A) vs. (Rab3A vs. ATG9A)], 1.826 [(Syph vs. ATG9A) vs. (Rab3A vs. ATG9A)].