Fig. 3

Loss of NHE9 dysregulates the pH of dendritic and axonal endosomes. a Dissociated hippocampal cultures prepared from NHE9 KO mice were co-transfected with HA-tagged mouse NHE9 and EGFP (to identify the transfected neurons). NHE9-HA colocalizes strongly with the transferrin receptor (TfR) in dendritic endosomes (Manders coefficient for TfR/HA 0.76 ± 0.07 s.e.m., Figure S3). In axons, NHE9-HA colocalizes with the vesicular glutamate transporter VGLUT1 at some but not all boutons (Manders coefficient for VGLUT1/HA 0.56 ± 0.04 s.e.m. Figure S3). Filled white arrowheads indicate colocalization and unfilled arrowheads HA-NHE9 alone. Scale bars all indicate 5 μm. b TfR-pHluorin was transfected into hippocampal neurons and imaged at DIV12–14. Dendritic endosomes expressing TfR-pHluorin exhibit a lower pH in NHE9 KO neurons than WT. ****p < 0.0001 by unpaired two-tailed Student’s t test. WT, n = 41 neurons/3 cultures; KO, n = 41 neurons/3 cultures. c Cytosolic pH was imaged in the soma and processes using the pH-sensitive ratiometric dye BCECF (2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein). A calibration curve for the dye performed in live neurons (Figure S4) was used to calculate pH. Bars indicate mean ± s.e.m. WT, n = 76 neurons/3 cultures; KO, n = 81 neurons/3 cultures. d VGLUT1-mOrange2 was transfected into hippocampal neurons and imaged at DIV12–14. Boutons containing VGLUT1-mOrange2 exhibit a lower average pH in NHE9 KO neurons than WT. *p < 0.05 by unpaired two-tailed Student’s t test. WT, n = 43 neurons/3 cultures; KO, n = 50 neurons/3 cultures (left panel). Frequency distribution (right panel) of average pH/ bouton for all boutons analyzed in the study, WT, n = 1415 boutons/3 cultures; KO, n = 1742 boutons/3 cultures. e Boutons expressing VAMP2-pHluorin exhibit a lower average lumenal pH in NHE9 KO neurons than WT. **p < 0.01 by unpaired two-tailed Student’s t test. WT, n = 40 neurons/3 cultures; KO, n = 41 neurons/3 cultures (left panel). Frequency distribution (right panel) of average pH/punctae for all punctae analyzed in the study, WT, n = 2330 boutons/3 cultures; KO, n = 1950 boutons/3 cultures