Fig 2: Rab7 GTP-GDP cycling is disrupted in NHE6-null rat brains.a, Schematic of GTP-GDP exchange cycle of Rab7. TBC1D5 is an GTPase activating protein (GAP) of Rab7. TBC1D5 promotes the hydrolysis of active, GTP-bound Rab7 to its inactive, GDP-bound form by stimulating GTP hydrolysis. Inactive Rab7 is then solubilized and extracted from membranes by GDP dissociation inhibitors (GDIs). Subsequently, guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP to reactivate Rab7.b, Representative images of fluorescence recovery after photobleaching (FRAP) assay with mEmerald-Rab7-transfected primary neurons at different time points. Slower recovery of mEmerald-Rab7 was detected from primary NHE6-null neurons compared to WT littermate neurons. Yellow circles indicate where photobleaching occurs and the fluorescence intensity was measured (WT = 4, Null = 4 pups, n = 35–37 neurons from four independent experiments). Non-linear mixed model was conducted. Scale bar, 5 μm. Scale bar for time course images, 0.1 μm.c, Reduced RILP-bound Rab7 (GTP-Rab7 pull-down) in NHE6-null rat brains at 2 months. Lysates from NHE6-null and WT littermate rat brains were incubated with His-RILP recombinant proteins or His-Myc (negative control) for the pull-down assay. Once samples were eluted, Rab7 was detected by western blot. Protein markers were loaded in the middle and labeled as “M”. The densitometry of GTP-Rab7 was divided by the total Rab7 for the quantification (4 rat brains for each genotype). Two-tailed unpaired t-test with Welch’s correction.d, Decreased GTP-bound Rab7 in NHE6-null rat brains at 2 months. Lysates from WT and NHE6-null littermate rat brains were incubated with GTP agarose to enrich GTP-bound protein fractions. Rab7 was detected in the GTP-enriched fraction by western blot. The densitometry of GTP-bound Rab7 was divided by the total Rab7 for the quantification (3 rat brains for each genotype). Two-tailed unpaired t-test with Welch’s correction was performed. Data are represented as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.

Fig 3: The interaction of NHE6 and TBC1D5 affects Rab7 GTPase activity.a, In vitro Rab7 GTPase assay shows that higher concentrations of TBC1D5 increased GTP hydrolysis of Rab7 measured by phosphate release. Pre-loaded GTP-Rab7 was incubated with various concentration of recombinant TBC1D5 for the luminescence measurement (n = 3 independent experiments). Ordinary one-way ANOVA with Dunnett’s test for multiple comparison was performed.b, Increased co-localization of Rab7 (green) and TBC1D5 (magenta) in NHE6-null neurons. The images were acquired using Nikon NSPARC. The quantifications show the Mander’s coefficient (M1: Rab7 overlapping TBC1D5, M2: TBC1D5 overlapping Rab7) from high-content imaging. Each dot indicates a mean of each pup (WT = 10, Null = 10 pups, n = 10,000–14,000 neurons imaged from each pup). Two-tailed unpaired t-test with Welch’s correction.c, Co-immunoprecipitation of NHE6 with TBC1D5 from WT and NHE6-null (negative control) rat brain lysates at 2 months shows the interaction of NHE6 with TBC1D5.d, Reciprocal immunoprecipitation from WT and NHE6-null littermate rat brain lysates at 2 months shows co-immunoprecipitation of TBC1D5 with NHE6.e, Representative images and the quantification of primary rat neurons show the co-localization of NHE6 (green) and TBC1D5 (magenta). Primary neurons were imaged using structured illumination microscopy (SIM). NHE6-null neurons were used as a negative control. A white box indicates the inset. The quantification presents Pearson’s correlation of NHE6 and TBC1D5 in WT and NHE6-null neurons. Means from each animal (big dots, WT = 3, Null = 3 pups) overlay the entire dataset (small dots, n = 13–15 neurons from three independent experiments) and used for statistical analysis. Two-tailed unpaired t-test with Welch’s correction was performed. Scale bar, 5 μm. Scale bar for the inset, 1 μm.f, In vitro Rab7 GTPase assay with increasing concentrations of NHE6. A constant concentration of GTP-Rab7 and TBC1D5 proteins (67.5 nM) was incubated with increasing concentration of NHE6 proteins to measure phosphate release. Higher concentrations of NHE6 decreased GTP hydrolysis of Rab7 mediated by TBC1D5. Ordinary one-way ANOVA with Tukey’s HSD. Three independent experiments were conducted. Data are represented as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.

Fig 4: Knock-down of the Rab7 GAP TBC1D5 rescues endosomal phenotypes in NHE6-null rat neurons.a, Reduced protein expression of TBC1D5 in primary littermate WT and NHE6-null rat neurons, with lentiviral transductions of anti-TBC1D5 shRNA, as compared to scrambled shRNA control transductions The expression of NHE6 and Rab7 was not affected by the shRNA transduction (three independent experiments). Two-way ANOVA with Fisher’s LSD test was performed.b, Representative kymographs from primary littermate WT and NHE6-null neurons with scrambled or TBC1D5 shRNA show the motility of mEmerald-Rab7 endosomes. Retrograde direction is indicated. Scale bar, 1 μm. The number of retrograde Rab7 endosomes in primary NHE6-null neurons transduced with TBC1D5 shRNA was increased compared to NHE6-null neurons transduced with scrambled shRNA. Also, the number of stationary Rab7 endosomes was decreased in primary NHE6-null neurons transduced with TBC1D5 shRNA. Means from each animal (big dots, WT = 3, Null = 3 pups) are overlay the entire dataset (small dots, n = 21–31 neurons from three independent experiments) and used for statistical analysis. Animals from same litters are color-coded. Ordinary one-way ANOVA with Tukey’s HSD was performed.c, The FRAP recovery rate of mEmerald-Rab7 in primary NHE6-null neurons transduced with TBC1D5 shRNA exhibited significant increase relative to NHE6-null neurons with scrambled shRNA. The recovery rate of mEmerald-Rab7 in NHE6-null neurons transduced with TBC1D5 shRNA was comparable to WT neurons (6 pups in total for each genotype, n = 20–35 neurons from 3 experiments). Non-linear mixed model was conducted.d, The increased endosomes-lysosomes fusion in primary NHE6-null neurons transduced with TBC1D5 shRNA. Shown are still images at different time points from live imaging of the endosome-lysosome fusion assay on primary littermate WT and NHE6-null neurons transduced with scrambled or TBC1D5 shRNA at different time points. The level of endosome-lysosome fusion is presented as the percentage fold change in overlap between the endosome label (magenta) and the lysosome label (green), from time point 0 for the same animal. Yellow arrows indicate the fusion events. (WT = 5, Null = 5 pups, n = 10,000–14,000 neurons imaged from each pup). Linear mixed model was conducted. Scale bar, 5 μm. Data are represented as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.

Fig 5: Rab7-positive late endosome defects in NHE6-null rats.a, Enlarged SMI32-stained (axonal damage marker, magenta) swellings in the corpus callosum (CC) and cortex (CTX) of NHE6-null rat brains and their littermate controls at 2 months. The number of SMI32-stained swellings were divided by the total number of SMI32 puncta. The average size of puncta was also quantified. Two-tailed unpaired t-test with Welch’s correction. Scale bar, 5 μm. (a-c) Means from each independent experiment (big dots, WT = 3, Null = 3 animals) overlay the entire dataset (small dots, 5 sections per each animal) and used for statistical analysis. Animals from same litters are color-coded.b, Accumulation of Rab7 late endosomes in the axons of NHE6-null rat cortex at 6 months. The percentage (%) of Rab7 area covered per image and within Tau-stained axons were quantified respectively. Two-tailed unpaired t-test with Welch’s correction. Scale bar, 20 μm.c, Enlarged Rab7 late endosomes in the CC and CTX of NHE6-null rats and their littermate controls at 2 months. White rectangles indicate magnified insets. The number of enlarged Rab7 (bigger than 1.5 μm) was divided by the total number of Rab7 puncta (%). The average size of Rab7 puncta was also measured. Two-tailed unpaired t-test with Welch’s correction. Scale bar, 10 μm.d, Representative snapshots and kymographs from WT and NHE6-null neurons show the motility of mEmerald-Rab7. Retrograde direction is indicated. Scale bar, 1 μm.e, Increased numbers of stationary mEmerald-Rab7 labelled endosomes in NHE6-null neurons. Decreased numbers of retrograde Rab7 endosomes were also detected in NHE6-null neurons. Anterograde, retrograde, and stationary endosomes were divided by the total number of mEmerald-Rab7 endosomes. Means from each animal (big dots, WT = 4, Null = 4 pups) overlay the entire dataset (small dots, n = 17–20 neurons from four independent experiments) and used for statistical analysis. Animals from same litters are color-coded. Ordinary one-way ANOVA with Tukey’s HSD. Data are represented as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.