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NHE6 depletion corrects ApoE4-mediated synaptic impairments and reduces amyloid plaque load

  1. Theresa Pohlkamp  Is a corresponding author
  2. Xunde Xian
  3. Connie H Wong
  4. Murat S Durakoglugil
  5. Gordon Chandler Werthmann
  6. Takaomi C Saido
  7. Bret M Evers
  8. Charles L White III
  9. Jade Connor
  10. Robert E Hammer
  11. Joachim Herz  Is a corresponding author
  1. Department of Molecular Genetics, University of Texas Southwestern Medical Center, United States
  2. Center for Translational Neurodegeneration Research, United States
  3. Institute of Cardiovascular Sciences and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Peking University, China
  4. Laboratory for Proteolytic Neuroscience, Riken Center for Brain Science, Japan
  5. Pathology, University of Texas Southwestern Medical Center, United States
  6. Department of Biochemistry, University of Texas Southwestern Medical Center, United States
  7. Department of Neuroscience, University of Texas Southwestern Medical Center, United States
  8. Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, United States
Research Article
Cite this article as: eLife 2021;10:e72034 doi: 10.7554/eLife.72034
10 figures, 1 table and 1 additional file

Figures

ApoE4 induces endolysosomal trafficking delay.

(A) pH regulation within the endolysosomal pathway. Upon receptor binding, ApoE is endocytosed along with glutamate receptors (AMPA and NMDA receptors). Cargo that has entered the early endosome (EE) can undergo recycling through a fast direct route without further acidification (fast recycling) or through slower sorting pathways that require further acidification (Casey et al., 2010; Naslavsky and Caplan, 2018). While lipid components are targeted to the lysosome, the majority of receptors, as well as ApoE, remain in endosomal compartments at the cellular periphery where they rapidly move back to the surface (Heeren et al., 1999). The increasingly acidic luminal pH is illustrated as a color gradient and depicted on the left. (B) In the presence of ApoE4, early endosomal trafficking and fast recycling are delayed. At the pH of the EE, ApoE4 is near its isoelectric point where solubility is reduced (Wintersteiner and Abramson, 1933), impairing receptor dissociation and resulting in delayed endosomal maturation with a concomitant entrapment of co-endocytosed glutamate receptors. Endosomal pH is regulated by the vesicular ATPase and the counterregulatory action of the proton leakage channel NHE6. NHE6 is an antiporter that exchanges a Na+ or K+ ion for each proton. As the pH decreases, ligands dissociate from their receptors allowing the EE to mature. If dissociation is delayed, as in case of ApoE4, endosomal trafficking is arrested, leading to progressive acidification as Na+, K+, and Cl- ions continue to enter the endosome to maintain charge neutrality while also drawing in water molecules due to osmotic pressure. We thus propose a model in which delayed ApoE4-receptor dissociation prevents early endosomal maturation and causes osmotic swelling while the pH continues to decrease until dissociation occurs. (C) Accelerated endosomal acidification by inhibition of the proton leak channel NHE6 resolves ApoE4 accumulation, promotes rapid receptor dissociation, and promotes the vesicle entry into the lysosomal delivery or recycling pathways.

Generation of Slc9a6fl and Slc9a6- mice.

(A) Gene targeting strategy. LoxP sites were introduced to flank the first exon (E1) of Slc9a6 (located on the X-chromosome) by gene targeting in embryonic stem cells. The targeting construct contained a long arm of homology (LA, gray) upstream of the first loxP site and the first exon. An loxP/FRT-flanked neomycin resistance cassette was cloned downstream of the first exon, followed by a short arm of homology (SA, gray). The targeted locus is shown below. Targeted stem cells were used to generate chimeric Slc9a6fl mice. Germline NHE6 knockout mice (NHE6-/- [female], NHE6y/- [male]; rec indicates recombined allele) were generated by breeding the Slc9a6fl line with Meox-Cre mice. (B) Genotyping of wildtype (wt, +), floxed (fl), and recombined (rec, -) NHE6 alleles. The PCR amplified regions are indicated in panel A. The wildtype and floxed allele PCR products differ by 50 bp (270 for floxed, 220 for wildtype). (C) Western blot showing brain lysates (left) of different NHE6 genotypes after Meox-Cre-induced germline recombination. (D) Mouse embryonic fibroblasts from Slc9a6- and control littermate were infected with Vamp3-pHluorin2 and excited at 408 and 488 nm with emission measured at 510 nm. (E) Vesicular pH measured using a standard curve was significantly decreased in Slc9a6- fibroblasts. Data is expressed as mean ± SEM. Statistical analysis was performed using Student’s t-test. (**p < 0.01) (F) The percent of vesicles with pH >6.4 is significantly decreased in Slc9a6- fibroblasts. (G) CAG-CreERT2 activity after tamoxifen application in a reporter mouse line expressing tdTomato. CreERT2 recombination activity without (left panel) or with (middle panel) tamoxifen application in the CAG-CreERT2 line bred with Rosa26floxStop-tdTomato line. After tamoxifen induction, CreERT2 activity led to a robust tdTomato signal in the hippocampus (middle panel). Pyramidal neurons in the CA1 pyramidal cell layer (PCL) (middle panel) are shown magnified in the right panel.

Long-term sodium-hydrogen exchanger 6 (NHE6) deficiency induced after Purkinje cell maturation causes Purkinje cell loss.

(A) Experimental timeline for B, mice were injected with tamoxifen at 2 months; after 1 month the brains were analyzed for NHE6 expression (Tam = tamoxifen, Exp. = experiment, mo. = months). (B) Western blot showing the efficiency of tamoxifen-induced NHE6 knockout in different brain regions (CA1, CA3, dentate gyrus, cortex, and cerebellum). The knockout efficiency differed between brain regions, it was 80% ± 2% in CA1, 82 ± 5.7% in the CA3, 67 ± 6.8% in the dentate gyrus, 65% ± 11.2% in the cortex, and 74% ± 4.7% in the cerebellum. A total of three brains in each group were examined. (C–F) NHE6 deficiency leads to cerebellar Purkinje cell loss in germline (Slc9a6-, C) and conditional (Slc9a6fl;CAG-CreERT2, D–F) knockout mice. Slc9a6+ includes both female wildtypes (Slc9a6+/+) and male wildtypes (Slc9a6y/+) mice. Slc9a6- includes both female knockouts (Slc9a6-/-) and male knockouts (Slc9a6y/-) mice. In addition, Slc9a6fl mice includes both female Slc9a6fl/fl and male Slc9a6y/fl. The timeline shows that Slc9a6fl;CAG-CreERT2 and control mice were tamoxifen-injected at 2 months and analyzed 1 year after (D). Calbindin was fluorescently labeled to highlight Purkinje cells in the cerebellum. Massive loss of Purkinje cells was found in Slc9a6- (C, lower panel), compared to wildtype Slc9a6+ control (C, upper panel). Long-term loss of NHE6, induced after Purkinje cell maturation at 2 months of age, also led to massive Purkinje cells loss when mice were examined 1 year after NHE6 ablation (E, lower panel). (F) Quantification of Purkinje cell loss in the cerebellum of Slc9a6fl;CAG-CreERT2 mice. Values are expressed as mean ± SEM from four independent experiments. Statistical analysis was performed using Student’s t-test. *p < 0.05.

Sodium-hydrogen exchanger 6 (NHE6) deficiency alleviates ApoE4-impaired surface trafficking deficits of Apoer2 and glutamate receptors.

(A) Timeline for the receptor surface expression assay applied for the experiments shown in B–F. Primary neurons were treated with naturally secreted ApoE3 or ApoE4 and/or Reelin before they underwent surface biotinylation. (B–F) Wildtype and Slc9a6- primary neurons were prepared from littermates and used in the receptor surface expression assay described in A. Slc9a6+ includes both female wildtypes (Slc9a6+/+) and male wildtypes (Slc9a6y/+) mice. Slc9a6- includes both female knockouts (Slc9a6-/-) and male knockouts (Slc9a6y/-) mice. (B) NHE6 deficiency was confirmed via Western blot, β-actin was used as loading control. (C–F) ApoE-conditioned media treatment reduces the surface expression of Apoer2 and glutamate receptors in presence of Reelin in primary neurons. Receptor surface levels show a stronger reduction with ApoE4 than ApoE3. NHE6 depletion counteracts the ApoE4-induced reduction of receptor surface expression. Cell surface biotinylation assay was performed for Apoer2 (C), GluN2B (D), GluA1, (E) and GluA2/3 (F). Total levels were analyzed by immunoblotting of whole cell lysates against the same antibodies. β-Actin was used as loading control. Quantitative analysis of immunoblot signals is shown in the lower panels (C–F). All data are expressed as mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Dunnett’s post hoc test (C–F).

Effect of conditional sodium-hydrogen exchanger 6 (NHE6) knockout on Reelin-potentiated synaptic plasticity.

(A–H) Conditional knockout of NHE6 restores Reelin-enhanced long-term potentiation (LTP) in ApoeAPOE4 mice. Reelin facilitated induction of LTP in ApoeAPOE3 (A, E), but not ApoeAPOE4 (C, G) control (Slc9a6fl) mice. Slc9a6 deficiency in ApoeAPOE3 mice caused a reduction in Reelin-enhanced LTP, such that it is not significantly different from the control LTP (B, F). Importantly, in ApoeAPOE4;Slc9a6fl;CAG-CreERT2 mice Reelin was able to enhance theta burst-induced potentiation (D, H). Hippocampal slices were prepared from 3-month-old double mutant mice with either human ApoeAPOE3 or ApoeAPOE4 crossed with Slc9a6 conditional knockout mice (Slc9a6fl;CAG-CreERT2, tamoxifen injections at 6–8 weeks). Extracellular field recordings were performed in slices treated with or without Reelin. Theta burst stimulation (TBS) was performed after 20 min of stable baseline. Representative traces are shown in each panel, before TBS induction (black) and 40 min after TBS (gray). (E–H) Quantitative analysis of normalized fEPSP slopes at time intervals as indicated. (I, J) Input output curves of ApoeAPOE3 (I) and ApoeAPOE4 (J) mice with or without Slc9a6fl;CAG-CreERT2. Slc9a6fl mice includes both female Slc9a6fl/fl and male Slc9a6y/fl mice. Apoe mice are homozygous for APOE3 or APOE4. All data are expressed as mean ± SEM. N-numbers for each genotype group and treatment are indicated in panels A–D. *p < 0.05. Statistical analysis was performed using Student’s t-test.

Na+/H+ exchanger (NHE) inhibition or sodium-hydrogen exchanger 6 (NHE6) knockdown does not alter β-site amyloid precursor protein cleaving enzyme 1 (BACE1) activity in primary neurons.

(A, B) Pan-NHE inhibition by EMD87580 or lentiviral knockdown of Slc9a6 (NHE6) did not alter BACE1 activity in primary neurons of AppSwe mice (Tg2576). (A) DIV10 primary neurons were treated with γ-secretase inhibitor L-685458, EMD87580, and/or ApoE4 (as indicated) and harvested for immunoblotting against Aβ-containing C-terminal fragment of APP (β-CTF). β-Actin was blotted as loading control. Bar graph shows the statistics of n = 3 experiments. (B) Primary neurons of AppSwe mice were infected with lentivirus for shRNA expression directed against Slc9a6(NHE6) (shSlc9a6) or a scramble control sequence (-) at DIV7. At DIV13 neurons were treated with L-685458 overnight and harvested for immunoblotting against NHE6 and β-CTF on DIV14. RAP was blotted as loading control. Bar graph shows the statistics of n = 6 experiments. All data are expressed as mean ± SEM. Statistical analysis was performed using Student’s t-test. n.s. = not significant.

Figure 7 with 3 supplements
Sodium-hydrogen exchanger 6 (NHE6) deficiency decreases plaque formation in both AppNL-F and AppNL-F;ApoeAPOE4 mice.

(A, B) NHE6-deficient AppNL-F and control AppNL-F mice were analyzed for plaque deposition at an age of 12 months. Thioflavin S staining was performed to visualize plaques. Plaques were found more frequently in the control AppNL-F mice (left panel in A), magnifications of the boxed areas are shown in the two middle panels. The plaque load between Slc9a6- mice and control littermates (all AppNL-F) was compared and analyzed. (B) In the Slc9a6- littermates, the plaque number was reduced, when compared to controls. (C–D) Slc9a6fl;CAG-CreERT2;AppNL-F;ApoeAPOE4 and Slc9a6fl;AppNL-F;ApoeAPOE4 mice were analyzed for plaque deposition. NHE6 was ablated at 2 months and brains were analyzed at 13.5–16 months. 4G8-immunolabeling against Aβ was performed to visualize plaques. In AppNL-F;ApoeAPOE4 mice conditional Slc9a6 knockout caused a reduction in plaque load compared to the Slc9a6fl control littermates. (C) Magnifications of the boxed areas in C are shown in the middle. (D) Plaque load was analyzed and compared between Slc9a6fl;CAG-CreERT2 mice and floxed control littermates. (I) Hematoxylin and eosin (H&E) staining was performed to investigate for gross anatomic abnormalities in the Slc9a6fl;CAG-CreERT;AppNL-F;ApoeAPOE4 and Slc9a6fl;AppNL-F;ApoeAPOE4 mice. (F–I) Brain area (F), cortical thickness (G), hippocampal (HC) area (H), and CA1 thickness (I) were analyzed. Student’s t-test did not reveal a significant difference. Plaques were differentiated by size or staining density as described in detail in the supplements (Figure 7—figure supplement 2). Labeled plaques were analyzed by a blinded observer. All data are expressed as mean ± SEM. (B) Slc9a6- n = 5, control n = 8, (C) Slc9a6fl n = 8 (Slc9a6fl;CAG-CreERT2 n = 8), in (F–I) derived from n = 5 (Slc9a6fl) and n = 6 (Slc9a6fl;CAG-CreERT2) animals. *p < 0.05. **p < 0.01, ***p < 0.005. Slc9a6+ represents both female wildtypes (Slc9a6+/+) and male wildtypes (Slc9a6y/+). Slc9a6- represents both female knockouts (Slc9a6-/-) and male knockouts (Slc9a6y/-). In addition, Slc9a6fl mice includes both female Slc9a6fl/fl and male Slc9a6y/fl mice. Apoe mice are homozygous for APOE4 (ApoeAPOE4). AppNL-F mice are homozygous for human NL-F knockin mutation (AppNL-F/NL-F). Statistical analysis was performed using two-way analysis of variance (ANOVA) with Sidak’s post hoc test (B and D) and Student’s t-test (F–I).

Figure 7—figure supplement 1
Gross anatomical brain structure in Slc9a6- mice.

(A) Hematoxylin and eosin (H&E) staining was performed to investigate for gross anatomic abnormalities in the Slc9a6-;AppNL-F and AppNL-F mice. Structures representing plaques were found in the AppNL-F control groups (magnified example is shown in the middle panel). (B–E) Brain area (B), cortical thickness (C), hippocampal (HC) area (D), and CA1 thickness (E) were analyzed. All data are expressed as mean ± SEM. Student’s t-test did not reveal significant differences, n = 3 (control) and n = 4 (Slc9a6-). Slc9a6+ represents both female wildtypes (Slc9a6+/+) and male wildtypes (Slc9a6y/+). Slc9a6- represents both female knockouts (Slc9a6-/-) and male knockouts (Slc9a6y/-). AppNL-F mice are homozygous for human NL-F knockin mutation (AppNL-F/NL-F).

Figure 7—figure supplement 2
Example of Thioflavin S stained plaques for quantification.

Different types of Thioflavin S stained plaques and 4G8-immunoreactive accumulations in AppNL-F brains are shown. Different sizes of plaques were grouped together for quantification Thioflavin S-labeled plaques (Figure 7 and Figure 7—figure supplement 3). Plaques bigger in diameter than 20 µm with a dense core were defined as big. Medium sized plaques had a diameter between 10 and 20 µm with a dense core. Small plaques were smaller than 10 µm and often represented individual cells. 4G8-labeled plaques were differentiated by diffuse or dense appearance as depicted in the examples.

Figure 7—figure supplement 3
Sodium-hydrogen exchanger 6 (NHE6) deficiency decreases plaque formation in both AppNL-F and AppNL-F;ApoeAPOE4 mice.

(A–B) NHE6-deficient AppNL-F and control AppNL-F mice were analyzed for plaque deposition at an age of 12 months. 4G8-immunolabeling against Aβ (A) visualized more plaques in the control AppNL-F mice. The plaque load between Slc9a6- mice and control littermates (all AppNL-F) was compared and analyzed. (B) In the Slc9a6- littermates the plaque number was reduced, when compared to controls. (C–E) Soluble (TBS) and insoluble (GuHCl and 70% FA) Aβ fractions of cortical lysates were analyzed by commercial ELISA; 1.5-year-old Slc9a6- mice showed less insoluble Aβ than their control littermates (all AppNL-F). (F–G) Slc9a6fl;CAG-CreERT2;AppNL-F;ApoeAPOE4 and Slc9a6fl;AppNL-F;ApoeAPOE4 mice were analyzed for plaque deposition. NHE6 was ablated at 2 months and brains were analyzed at 13.5–16 months. Thioflavin S staining was performed to visualize plaques. With AppNL-F;ApoeAPOE4 background, the Slc9a6fl;CAG-CreERT2 mice had a reduced plaque load compared to the Slc9a6fl control mice (left panel in F). Magnifications of the boxed areas in left panel are shown in the middle. (G) Plaque load was analyzed and compared between Slc9a6fl;CAG-CreERT2 mice and floxed control littermates. Plaques were differentiated by size or staining density as described in detail in the supplements (Figure 7—figure supplement 2). Labeled plaques were analyzed by a blinded observer (B, G). All data (immunohistochemistry: Slc9a6- n = 5, control n = 4, Slc9a6fl n = 10, Slc9a6fl;CAG-CreERT2 n = 12; biochemistry: Slc9a6- n = 11, control n = 7) are expressed as mean ± SEM. *p < 0.05. **p < 0.01, ***p < 0.005. Slc9a6+ represents both female wildtypes (Slc9a6+/+) and male wildtypes (Slc9a6y/+). Slc9a6- represents both female knockouts (Slc9a6-/-) and male knockouts (Slc9a6y/-). In addition, Slc9a6fl mice includes both female Slc9a6fl/fl and male Slc9a6y/fl mice. Apoe mice are homozygous for APOE4. AppNL-F mice are homozygous for human NL-F knockin mutation (AppNL-F/NL-F). Statistical analysis was performed using two-way analysis of variance (ANOVA) with Sidak’s post hoc test.

Age-dependent increase in plaque load is abolished in Slc9a6fl;CAG-CreERT2;AppNL-F;ApoeAPOE4 mice.

(A–E) Slc9a6fl;CAG-CreERT2;AppNL-F;ApoeAPOE4 and Slc9a6fl;AppNL-F;ApoeAPOE4 mice were analyzed for plaque deposition. Sodium-hydrogen exchanger 6 (NHE6) was ablated at 2 months and brains were analyzed at 13.5–16 months. 4G8-immunolabeling against Aβ (A,B) and Thioflavin S staining (C–E) were performed to visualize plaques (Figure 7C and Figure 7—figure supplement 3F). Plaque load was analyzed and compared between Slc9a6fl;CAG-CreERT2 mice and floxed control littermates. Plaques were differentiated by staining intensity (A, B) or size (C–E) as described in the supplements (Figure 7—figure supplement 2). In the time range analyzed, plaque load increased by age in control, but not in Slc9a6fl;CAG-CreERT2 mice. Plaques were analyzed by a blinded observer. Plaque count (Slc9a6fl;CAG-CreERT2 n = 8, Slc9a6fl n = 8 for A) (B); Slc9a6fl;CAG-CreERT2 n = 12; Slc9a6fl n = 10 in (C–E) is pis plotted against age of mice. Slc9a6fl mice includes both female Slc9a6fl/fl and male Slc9a6y/fl mice. Apoe mice are homozygous for APOE4. AppNL-F mice are homozygous for human NL-F knockin mutation (AppNL-F/NL-F).

Figure 9 with 2 supplements
Microglia and astrocytes surround plaques in both AppNL-F control and AppNL-F;Slc9a6- brains.

(A–B) Co-labeling of microglia (Iba1, green, A) or astrocytes (GFAP, green, B) with Aβ (6E10, red) in brain slices of AppNL-F and AppNL-F;Slc9a6- mice. (C) Quantification of plaques in control and Slc9a6- brain slices. (D) Bar graph showing the intensity density of Iba1/6E10 as quantitative measure of microglia surrounding plaques. (E) Statistical analysis of 6E10 positive microglia and (F) the intensity of 6E10 signal within microglia. (G) Bar graph showing the intensity density of GFAP/Aβ as quantitative measure of astrocytes surrounding plaques. Data were analyzed by a blinded observer. All data are expressed as mean ± SEM. Data were obtained from n = 4 (control) and n = 5 (Slc9a6-) mice (A–G).(D) n = 33 (control) and n = 23 (Slc9a6-) plaques were analyzed, in (E) n = 22 (control) and n = 24 (Slc9a6-) microscopical pictures were analyzed, in (F) n = 31 (control) and n = 38 (Slc9a6-) 6E10 positive (defined as signal intensity above 500) microglia were analyzed, in (G) n = 33 (control) and n = 18 (Slc9a6-) plaques were analyzed. Student’s t-test revealed a difference in C (**p < 0.01) and did not reveal significant differences in (D–G). Slc9a6+ represents both female wildtypes (Slc9a6+/+) and male wildtypes (Slc9a6y/+). Slc9a6- represents both female knockouts (Slc9a6-/-) and male knockouts (Slc9a6y/-). AppNL-F mice are homozygous for human NL-F knockin mutation (AppNL-F/NL-F).

Figure 9—figure supplement 1
Sodium-hydrogen exchanger 6 (NHE6) deficiency causes an increase in Iba1 and glial fibrillary acidic protein (GFAP) immunoreactivity in both AppNL-F and AppNL-F;ApoeAPOE4 mice.

(A) Immunohistochemistry against GFAP and ionized calcium-binding adapter molecule (Iba1) was performed on brain slices obtained from 1-year-old AppNL-F mice deficient for NHE6 (AppNL-F;Slc9a6-) and control littermates (AppNL-F). GFAP (upper panels) and Iba1 (lower panels) immunoreactivity was increased in NHE6-deficient AppNL-F mice when compared to NHE6 expressing controls. (B) Slc9a6fl;CAG-CreERT2;AppNL-F;ApoeAPOE4 and Slc9a6fl;AppNL-F;ApoeAPOE4 mice were analyzed for immunoreactivity GFAP and Iba1. Mice were injected with tamoxifen at 2 months and brain slices obtained from 13.5- to 16-month-old mice. GFAP (upper panels) and Iba1 (lower panels) immunoreactivity was increased in NHE6-deficient AppNL-F;ApoeAPOE4 mice when compared to NHE6-expressing controls. (C–F) Intensity of the staining in various areas was compared for GFAP (C) and Iba1 (D) in AppNL-F;Slc9a6- and AppNL-F mice. Intensity of the staining for GFAP (E) and Iba1 (F) in Slc9a6fl;CAG-CreERT2;AppNL-F;ApoeAPOE4 and Slc9a6fl;AppNL-F;ApoeAPOE4 mice. Analysis was performed by a blinded observer. ‘White matter’ comprises corpus callosum, cingulum, and external capsule. All data (Slc9a6- n = 5; control n = 4; Slc9a6fl;CAG-CreERT2 n = 6; Slc9a6fl n = 8) are expressed as mean ± SEM. *p < 0.05. **p < 0.01, ***p < 0.005. Slc9a6+ represents both female wildtypes (Slc9a6+/+) and male wildtypes (Slc9a6y/+). Slc9a6- represents both female knockouts (Slc9a6-/-) and male knockouts (Slc9a6y/-). In addition, Slc9a6fl mice includes both female Slc9a6fl/fl and male Slc9a6y/fl mice. For the Cag-CreERT2, Cre+ + the absence of Cre (Slc9a6fl alone) and Cag-CreERT2 indicates the presence of Cre (Slc9a6fl;CAG-CreERT2), which are both injected with Tamoxifen at 2 months of age. Apoe mice are homozygous for APOE4. AppNL-F mice are homozygous for human NL-F knockin mutation (AppNL-F/NL-F). Statistical analysis was performed using Student’s t-test.

Figure 9—figure supplement 2
Examples of plaques surrounded by microglia and astrocytes.

(A) Co-labeling of microglia (Iba1, green) and Aβ (6E10, red) in brain slices of AppNL-F and AppNL-F;Slc9a6- mice. (B) Co-labeling of astrocytes (glial fibrillary acidic protein [GFAP], green) and Aβ (6E10, red) in brain slices of AppNL-F and AppNL-F; Slc9a6- mice. Slc9a6+ represents both female wildtypes (Slc9a6+/+) and male wildtypes (Slc9a6y/+). Slc9a6- represents both female knockouts (Slc9a6-/-) and male knockouts (Slc9a6y/-). AppNL-F mice are homozygous for human NL-F knockin mutation (AppNL-F/NL-F).

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Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)Mouse/Slc9a6flThis study
Refer to Materials and methods section for detailed description of mouse model production
Strain, strain background (Mus musculus)Mouse/Slc9a6-This study
Refer to Materials and methods section for detailed description of mouse model production
Strain, strain background (Mus musculus)Mouse/ApoeAPOE3Sullivan et al., 1997IMSR_TAC:2,542ApoeAPOE3
Strain, strain background (Mus musculus)Mouse/ApoeAPOE4Knouff et al., 1999IMSR_TAC:3,518ApoeAPOE4
Strain, strain background (Mus musculus)Mouse/B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/JThe Jackson Laboratory Madisen et al., 2010JAX #007909ROSAfloxedStop-tdTomato
Strain, strain background (Mus musculus)Mouse/CAG-cre/Esr15Amc/JThe Jackson Laboratory Hayashi and McMahon, 2002JAX #004682CAG-CreERT2
Strain, strain background (Mus musculus)Mouse/B6.129S4-Meox2tm1(cre)Sor/JThe Jackson Laboratory Tallquist and Soriano, 2000JAX 003755Meox-Cre
Strain, strain background (Mus musculus)AppNL-FSaito et al., 2014
AppNL-F
Strain, strain background (Mus musculus)Tg2576Charles River Hsiao et al., 1996Charles River Tg2576Tg2576, APPSwe
Strain, strain background (Rattus norvegicus)SD ratCharles RiverSC:400
Cell line (Homo sapiens)HEK293Thermo FisherR70507, RRID:CVCL_0045
Cell line (Homo sapiens)HEK293-TATCCCRL-3216
Cell line (Mus musculus)Neuro-2aATCCCCL-131
Cell line (Mus musculus)NHE6-KO (Slc9a6-) mouse embryonic fibroblasts (MEFs)This study
Refer to Materials and methods section for detailed description of MEF production
Cell line (Mus musculus)Slc9a6+ MEFs (Slc9a6- littermate)This study
Refer to Materials and methods section for detailed description of MEF production
AntibodyAnti-Aβ (clone 6E10) (mouse monoclonal)CovanceSIG-39320 RRID:AB_662798WB and IHC (1:1000)
AntibodyAnti-Aβ (clone 4 G8) (mouse monoclonal)CovanceSIG-39220 RRID:AB_10175152IHC (1:1000)
AntibodyAnti-phospho tyrosine (clone 4 G10) (mouse monoclonal)EMD MilliporeMillipore Cat# 05–321, RRID:AB_309678WB (1:1000)
AntibodyAnti-Apoer2 (rabbit polyclonal)Herz Lab, #2561, Trommsdorff et al., 1999
WB (1:1000)
AntibodyAnti-β-Actin (rabbit polyclonal)AbcamAb8227, RRID:AB_23051 86WB (1:3000)
AntibodyAnti-Calbindin D-28k (mouse monoclonal)SwantSwant Cat# 300, RRID:AB_10000347IHC (1:1000)
AntibodyAnti-GFAP (rabbit polyclonal)AbcamAbcam Cat# ab7260, RRID:AB_305808IHC (1:2000)
AntibodyAnti-GluA1 (rabbit polyclonal)Abcamab31232, RRID:AB_2113447WB (1:1000)
AntibodyAnti-GluA2/3 (rabbit polyclonal)EMD Millipore07–598, RRID:AB_31074 1WB (1:1000)
AntibodyAnti-GluN2B (rabbit polyclonal)Cell Signaling Technology4,207 S, RRID:AB_12642 23WB (1:1000)
AntibodyAnti-Iba1 (rabbit polyclonal)Wako019–19741, RRID:AB_839504IHC (1:1000)
AntibodyAnti-NHE6 (C-terminus) (rabbit polyclonal)Herz Lab, Xian et al., 2018
WB (1:1000)
AntibodyAnti-mouse-IgG AF594 (goat polyclonal)Thermo FisherA-11032, RRID:AB_2534091IHC (1:500)
AntibodyAnti-rabbit-IgG AF488 (goat polyclonal)Thermo FisherA-11034, RRID:AB_2576217IHC (1:500)
Commercial assay or kitAnti-mouse-IgG staining kitVectorMP-7602, RRID:AB_2336532
Commercial assay or kitAnti-rabbit-IgG staining kitVectorMP-7601, RRID:AB_2336533
Chemical compound, drugAntigen retrieval citrate bufferBioGenex, CatHK086-9K
Chemical compound, drugB-27 Supplement (50×), serum freeThermo Fisher17504044
Chemical compound, drugCytoseal 60Thermo Fisher8310
Chemical compound, drugDMEMSigma-AldrichD6046
Chemical compound, drugFuGENEPromegaE2311
Chemical compound, drugHBSS (1×)Gibco14175
Chemical compound, drugL-Glutamic acid (glutamate)Sigma-AldrichG1251
Chemical compound, drugγ-Secretase inhibitor L-685458Tocris Bioscience2627
Chemical compound, drugPenicillin-streptomycin solution, 100×Corning30–002 CI
Chemical compound, drugNeurobasal Medium (1×) liquid without Phenol RedThermo Fisher12348017
Chemical compound, drugNeutrAvidin AgaroseThermo Fisher29201
Chemical compound, drugNonidet P-40 AlternativeEMD Millipore492016
Chemical compound, drug32% Paraformaldehyde AQ solutionFisher Scientific15714 S
Chemical compound, drugPBS (1×)Sigma-AldrichD8537
Chemical compound, drugPenisillin-streptomycinCorning30–002 CI
Chemical compound, drugPhosphatase inhibitor cocktailThermo Fisher78420
Chemical compound, drugPoly-D-lysineSigma-AldrichA-003-M
Chemical compound, drugProtein A-Sepharose 4BThermo Fisher101042
Chemical compound, drugProteinase Inhibitor CocktailSigma-AldrichP8340
Chemical compound, drugSulfo-NHS-SS-biotinPierce21331
Chemical compound, drugTriton X-100Sigma-AldrichCAS9002-93-1
Chemical compound, drugTween 20SigmaP1379
OtherVectashield with DAPIVector LabsH-1200(DAPI 1.5 µg/ml)
Transfected construct (Mus musculus)pCrl, Reelin expression vectorD’Arcangelo et al., 1997N/A
Transfected construct (Homo sapiens)pcDNA3.1-ApoE3Chen et al., 2010N/AProgenitor pcDNA3.1-Zeo
Transfected construct (Homo sapiens)pcDNA3.1-ApoE4Chen et al., 2010N/AProgenitor pcDNA3.1-Zeo
Transfected construct (Mus musculus)pLKO.1 scramble shRNAXian et al., 2018N/A
Transfected construct (Mus musculus)pLKO.1 shNHE6Open Biosystem
Xian et al., 2018
TRCN0000068828Refer to shNHE6-a
Transfected construct (Mus musculus)psPAX2Addgene12260Plasmid was a gift
from Didier Trono
Transfected construct (Mus musculus)pMD2.GAddgene12259Plasmid was a gift
from Didier Trono
Transfected construct (Mus musculus)pJB-NHE6
targeting vector
This studyN/ARefer to Materials and methods
section for detailed
description
Recombinant DNA reagentpJB1 (plasmid)Braybrooke et al., 2000N/A
Recombinant DNA reagentpCR4-TOPO (plasmid)Thermo FisherK457502
Recombinant DNA reagentpLVCMVfull (plasmid)Xian et al., 2018N/A
Recombinant DNA reagentpME (plasmid)Stawicki et al., 2014Addgene #73794Plasmid was a gift
from David Raible
Recombinant DNA reagentpLVCMV Vamp3pHluorin2 (plasmid)This studyN/ARefer to Materials and methods
section for detailed
description
Recombinant DNA reagentBAC containing murine NHE6 sequence
(bacterial artificial chromosome)
BACPAC
Resources Center
RP23 364 F14
Software, algorithmAdobe Creative CloudAdobeRRID:SCR_010279
Software, algorithmGraphPad Prism 7.0GraphPad SoftwareRRID:SCR_002798
Software, algorithmFiji/ImageJNIHRRID:SCR_002285
Software, algorithmLabView7.0National InstrumentsRRID:SCR_014325
Software, algorithmNDP.view2Hamamatsu Photonics

Software, algorithmOdyssey
Imaging System
LI-CORRRID:SCR_014579
Software, algorithmClustal OmegaEMBL-EBIRRID:SCR_001591
Software, algorithmLeica TCS SPELeicaRRID:SCR_002140
Sequence-based reagentSA forwardIDT
GGATCCGTGT
GTGTGTTGGG
GGAGGGA
Sequence-based reagentSA reverseIntegrated DNA Technology
CTCGAGCTCAC
AATCAGCCCTTT
AAATATGCC
Sequence-based reagentGAP repair US forwardIntegrated DNA Technology
AAGCTTGCGGCC
GCTTCAATTTCTG
TCCTTGCTACTG
Sequence-based reagentGAP repair
US reverse
Integrated DNA Technology
AGATCTCAAGAA
AGTTAGCTAGA
AGTGTGTC
Sequence-based reagentGAP repair
DS forward
Integrated DNA Technology
AGATCTGTAGA
GGATGTGGGA
AAGAGAG
Sequence-based reagentGAP repair
DS reverse
Integrated DNA Technology
GTCGACGCGG
CCGACACACA
CAGATAAATAA
CCTCAAAAG
Sequence-based reagent5’ flanking 1st LoxP fragment forwardIntegrated DNA Technology
GCTTCTCTCG
AGCAAGAGTCAAC
Sequence-based reagent5’ flanking 1st LoxP fragment reverseIntegrated DNA Technology
GATATCAGCA
GGTACCACCAA
GATCTCAACCT
TATTGTCCTATA
TGCACAAAC
Sequence-based reagent3’ flanking 1st LoxP fragment forwardIntegrated DNA Technology
GTCTTGTTGGTA
CCTGATGAAATG
GACTACCTCCACTTG
Sequence-based reagent3’ flanking 1st LoxP fragment reverseIntegrated DNA Technology
ATCGATCTTCA
TAACCCATCTGGATA
Sequence-based reagentLoxP Oligo forwardIntegrated DNA Technology
GATCTGCTCAGC
ATAACTTCGTATAG
CATACATTATACG
AAGTTATGGTAC
Sequence-based reagentLoxP Oligo reverseIntegrated DNA Technology
CATAACTTCGTA
TAATGTATGCTAT
ACGAAGTTATGC
TGAGCAGATC
Sequence-based reagentGenotyping NHE6-floxed and wt forwardIntegrated DNA Technology
GAGGAAGC
AAAGTGTCA
GCTCC
Sequence-based reagentGenotyping NHE6-floxed and wt reverseIntegrated DNA Technology
CTAATCCCCTC
GGATGCTGCTC
Sequence-based reagentGenotyping NHE6-KO forwardIntegrated DNA Technology
GAGGAAGC
AAAGTGTCA
GCTCC
Sequence-based reagentGenotyping NHE6-KO reverseIntegrated DNA Technology
CCTCACAAGACT
AGAGAAATGGTTC
Sequence-based reagentVamp3 forwardIntegrated DNA Technology
TTCAAGCTTCAC
CATGTCTACAGG
TGTGCCTTCGGGGTC
Sequence-based reagentVamp3 reverseIntegrated DNA Technology
CATTGTCATCAT
CATCATCGTGTG
GTGTGTCTCTAA
GCTGAGCAACAG
CGCCGTGGACGGC
ACCGCCGGCCCCG
GCAGCATCGCCAC
CAAGCTTAAC
Sequence-based reagentpHluorin2 forwardIntegrated DNA Technology
CCGGTCCCAAGCTT
ATGGTGAGCAAGG
GCGAGGAGCTGTTC
Sequence-based reagentpHluorin2 reverseIntegrated DNA Technology
GCCCTCTTCTAGAG
AATTCACTTGTACAG
CTCGTCCATGCCGTG

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