Fig 2: S1pr expression in the enteric nervous system. (A) RT-PCR was used to determine mRNA expression of S1P receptors in a murine enteric neuronal cell line (EN). As positive controls, the lung (L) was used for S1pr1 and S1pr3, the spleen (S) for S1pr4, and the brain (B) for S1pr5 and Tubb3, which served as a marker control for neuronal origin genes. (B) Expression of S1P receptors was determined in a rat enteric glial cell line (EG). Gfap was used as a control marker gene. (C) RT-PCR results were verified by qRT-PCR in EN and rat EG cell lines. Fold change expression was compared with the marker gene expression of Tubb3 and Gfap. (D) Murine ENS primary cell cultures were stained for S1PR1, S1PR3 or S1PR5 in combination with the corresponding cell type markers GFAP or ßIII-tubulin. Scale bars represent 100 µm. S1PR = sphingosine-1-phosphate receptor. EN = enteric neurons, L = lung, S = spleen, B = brain, EG = enteric glial cells, GFAP = glial fibrillary acidic protein, RT-PCR = reverse transcription polymerase chain reaction, qRT-PCR = quantitative real-time polymerase chain reaction.

Fig 3: CREB3L2-ATF4 activation recapitulates transcription misregulation profile of AD retromer.(A) Retromer regulatory inputs within the CREB3L2-ATF4 transcription network. Pink-colored squares denote significant enrichments. (B) VPS35 ChIP-seq tracks alongside ENCODE-produced H3K27Ac marks and DNase I hypersensitivity profiles. (C) Retromer transcriptomic profiles, mined from Zhang et al. (4), in nondemented control and AD individuals (bulk prefrontal cortex). Data are log2-transformed. Rows denote individual cases (controls, n = 101; AD, n = 129). EHD1: Pearson r = 0.79; VPS29: Pearson r = -0.72; SNX1: Pearson r = 0.70; SNX6: Pearson r = 0.68; VPS35: Pearson r = -0.67; VPS26B: Pearson r = -0.65. (D) VPS35 (x axis) and CREB3L2 (y axis) mRNA expression prefrontal cortex. Controls, Pearson r = -0.4481, ***P < 0.0001; AD, Pearson r = -0.5595, ***P < 0.0001. (E) Retromer protein levels in hippocampal neurons infected with control or Creb3l2-targeting shRNAs. Measurements were normalized to ßIII-tubulin and are presented relative to control baseline. Mean ± SEM of n = 3 to 4 replicates; unpaired t tests. (F) ChIP-qPCR analysis of CREB3L2 normalized to total input chromatin. Mean ± SEM of n = 3 to 5 replicates; unpaired t tests. (G) RT-qPCR analysis of retromer gene expression in CREB3L2-ATF4 (C-A), CREB3L2-CREB3L2 (C-C), and control (L-L) hippocampal neurons. Measurements were normalized to Tubb3 (ßIII-tubulin) and are presented relative to control (L-L background). Plots show individual measurements and mean ± SEM of n = 5 to 7 replicates; one-way ANOVA with Tukey’s multiple comparison tests. (H) Western blot analysis of retromer protein levels in CREB3L2-ATF4 and CREB3L2-CREB3L2 neurons. Measurements were normalized to ßIII-tubulin levels and are shown relative to control (L-L) baseline. Plots show individual measurements and mean ± SEM of n = 4 replicates (Vps29, n = 3); one-way ANOVA tests with Sidak’s multiple comparison correction.