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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
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Cite this article as: eLife 2021;10:e72034 doi: 10.7554/eLife.72034

Abstract

Apolipoprotein E4 (ApoE4) is the most important and prevalent risk factor for late-onset Alzheimer’s disease (AD). The isoelectric point of ApoE4 matches the pH of the early endosome (EE), causing its delayed dissociation from ApoE receptors and hence impaired endolysosomal trafficking, disruption of synaptic homeostasis, and reduced amyloid clearance. We have shown that enhancing endosomal acidification by inhibiting the EE-specific sodium-hydrogen exchanger 6 (NHE6) restores vesicular trafficking and normalizes synaptic homeostasis. Remarkably and unexpectedly, loss of NHE6 (encoded by the gene Slc9a6) in mice effectively suppressed amyloid deposition even in the absence of ApoE4, suggesting that accelerated acidification of EEs caused by the absence of NHE6 occludes the effect of ApoE on amyloid plaque formation. NHE6 suppression or inhibition may thus be a universal, ApoE-independent approach to prevent amyloid buildup in the brain. These findings suggest a novel therapeutic approach for the prevention of AD by which partial NHE6 inhibition reverses the ApoE4-induced endolysosomal trafficking defect and reduces plaque load.

Introduction

ApoE is the principal lipid transport protein in the brain. Three different ApoE isoforms are common in humans: ApoE2 (ɛ2), ApoE3 (ɛ3), and ApoE4 (ɛ4). Each ApoE4 allele reduces the age of Alzheimer’s disease (AD) onset by approximately 3–5 years compared to ApoE3 homozygotes, which comprise ~80% of the human population (Roses, 1994; Sando et al., 2008). By contrast and by comparison, ApoE2 is protective against AD (Corder et al., 1994; Panza et al., 2000; West et al., 1994). ApoE is an arginine-rich protein and a major component of very-low-density lipoproteins (Shore and Shore, 1973). The number of positively charged arginine residues differs between the three human isoforms due to two single nucleotide polymorphisms in the ApoE gene. The most common isoform, ApoE3, has a charge neutral cysteine at amino acid position 112 and an arginine at position 158. The second most common isoform, ApoE4, has two arginines, while the less frequent ApoE2 has two cysteines at these respective positions. The positively charged arginines raise the net charge and thus the isoelectric point (IEP) of the protein (Eto et al., 1985; Warnick et al., 1979). The IEP of ApoE2 is the lowest (5.9), ApoE3 has an intermediate IEP of 6.1, and the IEP of ApoE4 is ~6.4 (Ordovas et al., 1987).

For cargo delivery, ApoE binds to lipoprotein receptors and undergoes endocytosis and recycling. Endocytic subcompartments become progressively more acidic, and the pH of these compartments is regulated by the opposing functions of vesicular ATP-dependent proton pumps (vATPase) and proton leakage channels (Na+/H+ exchangers, NHEs). Early endocytic vesicles are slightly acidic (pH, ~6.4), which facilitates ligand/receptor dissociation. Lysosomes are highly acidic (pH 4–5), which is required for the digestion of endocytosed biomolecules (Figure 1A; Casey et al., 2010; Naslavsky and Caplan, 2018). For maturation of the early endosomes (EEs) and entry into the next sorting stage, ligand/receptor dissociation is required. The pH-dependent release of ApoE from its receptor in the EE is important for endosomal maturation and cargo delivery (Yamamoto et al., 2008) for the ability of endosomal content to rapidly recycle to the cell surface (Heeren et al., 1999; Nixon, 2017). The early endosomal pH, which triggers ligand-receptor dissociation, closely matches the IEP of ApoE4 (Xian et al., 2018). Loss of net surface charge at the IEP is accompanied by reduced solubility in an aqueous environment, leading to impaired dissociation of ApoE4 from its receptors (Xian et al., 2018) and aided by a greater propensity of ApoE4 to form a molten globule configuration under acidic conditions (Morrow et al., 2002). Dysregulation of endolysosomal trafficking by ApoE4 causes an age-dependent increase in EE number and size (Nuriel et al., 2017).

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.

Based on these observations, we have proposed a model in which destabilization of ApoE4 in the acidic EE environment, combined with a greater propensity for self-association, results in delayed detachment from its receptors (Figure 1B). Subsequent endosomal swelling through K+, Na+, and H2O influx further impairs cargo delivery, receptor recycling, and ligand re-secretion. Importantly, in neurons, ApoE and its receptor Apoer2 travel together with glutamate receptors through the endosomal recycling pathway (Chen et al., 2010; Xian et al., 2018). Rapid endocytosis and subsequent recycling of synaptic receptors is triggered by the synaptic homeostatic modulator and Apoer2 ligand Reelin (Hiesberger et al., 1999; Trommsdorff et al., 1999). We previously showed that ApoE4, in contrast to ApoE3 and ApoE2, prevents Reelin-mediated glutamate receptor insertion at the synapse, a state we refer to as ApoE4-mediated Reelin resistance (Chen et al., 2010; Durakoglugil et al., 2009; Lane-Donovan and Herz, 2017; Lane-Donovan et al., 2014; Xian et al., 2018). Reduction of endosomal pH and increasing the differential to the ApoE4 IEP abolishes this effect in vitro (Xian et al., 2018).

The pH of EE compartments is controlled by the vATPase-dependent proton pump and proton leakage through NHE6 (Nakamura et al., 2005; Basu et al., 1976; Davis et al., 1987; Rudenko et al., 2002). NHE6 is encoded by the gene Slc9a6. We showed that EE acidification by pharmaceutical pan-NHE inhibition or selective NHE6/Slc9a6 knockdown in neurons prevents the ApoE4-caused trafficking delay of ApoE and glutamate receptors (Xian et al., 2018). NHE6 deficiency in humans causes neurodevelopmental defects, which result in Christianson syndrome, an X-linked genetic disorder characterized by cognitive dysfunction, autism, ataxia, and epilepsy. However, some Slc9a6 mutant variants causing Christianson syndrome in humans do not significantly alter the ion exchange properties of NHE6 (Ilie et al., 2020; Ilie et al., 2019) suggesting that Christianson syndrome could be caused by loss of NHE6 scaffolding functions and not by loss of endosomal pH regulation. To investigate whether NHE6 depletion can reverse ApoE4 pathology in vivo, we generated a conditional Slc9a6 knockout (KO) mouse line (Slc9a6fl;CAG-CreERT2) to avoid complications caused by neurodevelopmental defects by temporally and spatially controlling NHE6 ablation. We show that genetic NHE6 ablation attenuates both, the ApoE4-induced Reelin resistance and impaired synaptic plasticity in ApoE4 targeted replacement (ApoeAPOE4) mice using hippocampal field recordings.

The pathological hallmarks of AD are extracellular aggregates of the amyloid β (Aβ) peptide and intracellular tangles of hyperphosphorylated tau protein. Processing of the transmembrane amyloid precursor protein (APP) at the β- and γ-sites leads to Aβ production. Aβ forms neurotoxic oligomers and accumulates in plaques. The β-site amyloid precursor protein cleaving enzyme 1 (BACE1) cleaves APP in its extracellular juxtamembrane domain to create a membrane-anchored C-terminal fragment (β-CTF) and a soluble extracellular APP domain (sAPPβ). β-CTF is further cleaved by the γ-secretase complex, which leads to the release of the Aβ peptide. APP and its secretases co-localize in endosomal compartments where cleavage can occur (Wang et al., 2018). It has further been reported that BACE1 activity is preferentially active in acidic environments (Shimizu et al., 2008). We therefore investigated whether NHE6 depletion alters BACE1 activity in neurons and whether NHE6 deficiency leads to changes in plaque deposition in vivo. We found that NHE6 inhibition or knockdown did not alter BACE1 activity, as judged by unchanged Aβ generation. By contrast, NHE6 ablation led to glial activation and decreased plaque load in ApoeAPOE4 (Sullivan et al., 1997) and AppNL-F (Saito et al., 2014) double knockin mice.

Results

NHE6 is required for postnatal Purkinje cell survival

NHE6 germline KO mice (Slc9a6-) and tamoxifen-inducible conditional NHE6 KO mice (Slc9a6fl;CAG-CreERT2) were generated as described in Materials and methods and Figure 2A–C. To validate early endosomal pH acidification by NHE6 deficiency, we isolated mouse embryonic fibroblasts from the Slc9a6- line and infected them with a Vamp3-pHluorin2 lentivirus expressing a fusion protein consisting of the endosomal Vamp3 protein and the ratiometric pH indicator pHluorin2 (Stawicki et al., 2014). We found a significantly reduced number of vesicles with pH 6.4 and above in Slc9a6- fibroblasts when compared to control (Figure 2D–F).

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.

To induce the conditional KO (cKO) of Slc9a6, tamoxifen was administered to Slc9a6fl;CAG-CreERT2 mice at 2 months (Figure 3A) and experiments were performed at the indicated time points. Slc9a6 KO efficiency in the brains of tamoxifen-injected Slc9a6fl;CAG-CreERT2 mice was 65–82% and varied between brain regions (Figure 3B). To further investigate CreERT2 activity upon tamoxifen injection, we bred the CAG-CreERT2 line with a stop-tdTomato reporter line in which a floxed stop-codon precedes the tdTomato start-codon. After tamoxifen injection, brains were examined for tdTomato expression. Without tamoxifen injection, tdTomato-expressing cells were almost absent in the hippocampus. Tamoxifen-induced recombination led to a broad expression of tdTomato in the hippocampus (Figure 2G).

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.

Individuals with Christianson syndrome and mice lacking NHE6 present with motor deficits due to a dramatic progressive loss of cerebellar Purkinje cells (Ouyang et al., 2013). We have reproduced the Purkinje cell loss in our germline Slc9a6- mice (Figure 3C). Next, we investigated whether Purkinje cell loss is the consequence of neurodevelopmental or neurodegenerative effects caused by loss of NHE6. Slc9a6 deficiency was induced at 2 months, after Purkinje cells had developed and matured. One year after Slc9a6 ablation, Purkinje cell loss was indistinguishable from that seen in the germline KO (Figure 3D–F). Therefore, Purkinje cell degeneration manifests itself postnatally and is not developmentally determined by the absence of NHE6. However, it is possible that loss of scaffolding functions and proper sorting, rather than dysregulation of endosomal pH, could be the main mechanism that causes Christianson syndrome, including Purkinje cell loss (Ilie et al., 2020; Ilie et al., 2019). If this could be substantiated by the development or discovery of Slc9a6 mutants that selectively ablate its ion exchange capacity without affecting its subcellular sorting or interaction with cytoplasmic or luminal binding partners, this would further raise the potential of NHE6 as a novel drug target for neurodegenerative diseases.

Genetic disruption of NHE6 restores trafficking of Apoer2, AMPA, and NMDA receptors in the presence of ApoE4

As we reported previously, ApoE4 impairs the trafficking of synaptic surface receptors (Chen et al., 2010). To monitor receptor recycling in neurons, we made use of an assay where Reelin is used to modulate receptor surface expression. Reelin is applied to primary neurons for 30 min in the presence or absence of naturally secreted, receptor-binding competent ApoE (Figure 4). Subsequently, surface biotinylation is performed and cells are harvested for immunoblotting to quantify the amount of Apoer2 and glutamate receptors expressed on the neuronal surface (Chen et al., 2010; Xian et al., 2018).

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).

We have shown previously that in the presence of ApoE4, Apoer2 and glutamate receptors recycle poorly to the neuronal surface. This recycling block could be resolved by endosomal acidification induced by shRNA knockdown of Slc9a6 or by applying the NHE inhibitor EMD87580 (Xian et al., 2018). To further exclude a nonspecific effect caused by the inhibition of other NHE family members or by shRNA off-target effects, we applied this assay on neurons isolated from Slc9a6- embryos (Figure 4B). Apoer2 recycling was completely restored in Slc9a6- neurons treated with ApoE4 (Figure 4C). We previously reported that the addition of ApoE3 to neurons also affects Apoer2 trafficking to a small, but reproducible extent. This was also abolished in Slc9a6- neurons (Figure 4C). In addition, genetic loss of NHE6 equally restored the ApoE4-impaired surface expression of AMPA and NMDA receptor subunits (Figure 4D–F).

Conditional disruption of NHE6 relieves synaptic Reelin resistance in ApoeAPOE4 mice

Reelin can enhance long-term potentiation (LTP) in hippocampal field recordings of ApoeAPOE3 but not ApoeAPOE4 acute brain slices (Chen et al., 2010). We previously showed that this Reelin resistance in ApoeAPOE4 slices was attenuated by pharmacological NHE inhibition (Xian et al., 2018). To investigate if endogenous loss of NHE6 also restores synaptic plasticity in the presence of ApoE4, we performed hippocampal field recordings on Slc9a6fl;CAG-CreERT2 mice bred to ApoeAPOE3 or ApoeAPOE4 mice. To avoid potentially compounding effects of NHE6 deficiency during embryonic development (Ouyang et al., 2013), NHE6/Slc9a6 gene disruption was induced at 8 weeks by intraperitoneal tamoxifen injection (Lane-Donovan et al., 2015). Electrophysiology was performed 3–4 weeks after NHE6 depletion in 3-month-old mice (Slc9a6fl;CAG-CreERT2, Figure 5B, D, F and H). Tamoxifen-injected Slc9a6fl;CAG-CreERT2-negative mice expressing human ApoE3 or ApoE4 served as controls (ApoeAPOE3 and ApoeAPOE4, Figure 5A, C, E and G). For field recordings, hippocampal slices were perfused with Reelin as described (Beffert et al., 2005; Chen et al., 2010; Durakoglugil et al., 2009; Weeber et al., 2002). Conditional genetic loss of NHE6 resulted in a moderate reduction of the ability of Reelin to enhance LTP in ApoeAPOE3 mice (comparing Figure 5A and E to B and F). By contrast, as reported previously (Chen et al., 2010), hippocampal slices from ApoeAPOE4 mice were completely resistant to LTP enhancement by Reelin (Figure 5C and G). This resistance was abolished when NHE6 was genetically disrupted in ApoeAPOE4 mice: Reelin application enhanced LTP (Figure 5D and H) in ApoeAPOE4;Slc9a6fl;CAG-CreERT2 to a comparable extent as in the ApoeAPOE3;Slc9a6fl;CAG-CreERT2 mice (Figure 5B). Synaptic transmission was monitored and input-output curves were generated by plotting the fiber volley amplitude, measured at increasing stimulus intensities, against the fEPSP slope. No significant differences were found (Figure 5I and J).

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.

NHE inhibition or NHE6 knockdown does not alter β-CTF generation in vitro

Cleavage of APP by γ-secretase and the BACE1 generates the short neurotoxic polypeptide Aβ. Cleavage by BACE1 results in a membrane anchored fragment called β-CTF, which is further processed by γ-secretase to yield the soluble Aβ peptide. BACE1 processing of APP occurs in the Golgi complex, on the cell membrane, and after endocytosis in endosomes (Caporaso et al., 1994; Vassar et al., 1999). It has been reported that BACE1 activity increases with lower pH (Hook et al., 2002). In a recent in vitro study in an HEK293 cell line overexpressing APP and BACE1, NHE6 overexpression reportedly led to a reduction of Aβ production and conversely shRNA knockdown of NHE6/Slc9a6 resulted in an increase in Aβ production (Prasad and Rao, 2015). To investigate if NHE6/Slc9a6 deficiency contributes to APP processing by BACE1 in neurons, we used primary neurons derived from AppSwe (Tg2576) mice, an Alzheimer’s disease (AD) mouse model that overexpresses human APP with the ‘Swedish’ mutation (Hsiao et al., 1996). Neurons were treated with the NHE inhibitor EMD87580 or transduced with lentiviral shRNA directed against NHE6/Slc9a6 in the presence or absence of the γ-secretase inhibitor L-685458. β-CTF was detected using the monoclonal antibody 6E10 directed against Aβ residues 1–16 (Figure 6). Inhibition of γ-secretase in ApoE4-treated AppSwe neurons strongly enhanced β-CTF accumulation, however, additional treatment with EMD87580 did not alter the amount of β-CTF in the cell lysates (Figure 6A). NHE6 knockdown using lentiviral shRNA also had no effect on the amount of β-CTF (Figure 6B). We conclude that NHE6 inhibition is unlikely to increase Aβ production under near-physiological conditions.

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.

NHE6 deficiency reduces Aβ plaque load in human App knockin mice

To further study the effect of NHE6 deficiency on Aβ pathology in vivo, we bred humanized AppNL-F mice (Saito et al., 2014) to our germline NHE6 KO line (Slc9a6-). In these AppNL-F mice, the Aβ sequence has been completely humanized and the early onset AD Swedish mutation (5’ located mutations encoding K670N and M671L = NL) and the Beyreuther/Iberian mutation (3’ flanking mutation encoding I716F = F) were also introduced, resulting in increased Aβ production, but physiological regulation of APP expression. This allowed us to determine the effect of Aβ overproduction while keeping APP expression under the control of the endogenous promoter. AppNL-F;Slc9a6- and control AppNL-F littermates were aged to 1 year. Perfusion-fixed brains were harvested and analyzed by hematoxylin and eosin (H&E) staining, Thioflavin S staining to visualize plaque load, and Aβ immunohistochemistry. H&E staining did not reveal any obvious anatomical structural differences between genotypes, but brain size, cortical thickness, hippocampal area, and CA1 thickness were reduced, as described previously (Xu et al., 2017; Figure 7—figure supplement 1). Plaques were more frequent in Slc9a6 wildtype than Slc9a6- mice. To further investigate and quantify plaque load, we analyzed the same brains after Thioflavin S staining (Figure 7A). We found an approximate 80% reduction of plaques in Slc9a6- mice when compared to littermate controls (Figure 7B). Immunohistochemistry against Aβ showed the same reduction (Figure 7—figure supplement 3A,B). In addition, we analyzed soluble (TBS fraction) and insoluble (GuHCl and 70 % FA fractions) Aβ in cortical brain lysates of 1.5-year-old AppNL-F;Slc9a6- mice and their control littermates. The amount of insoluble Aβ (GuHCl and 70% FA fractions) was reduced in NHE6-depleted mice by approximately 71%, when compared to their control littermates (Figure 7—figure supplement 3C-E). The ~50% reduction in soluble Aβ was statistically not significant in Slc9a6- lysates (TBS fraction).

Figure 7 with 3 supplements see all
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).

NHE6/Slc9a6 deficiency reduces plaque load in AppNL-F;ApoeAPOE4

To further investigate whether Slc9a6 deficiency also protects the brain from plaques in the presence of human ApoE4 instead of murine ApoE, we bred Slc9a6fl;CAG-CreERT2; ApoeAPOE4 with AppNL-F mice. At 2 months of age, we induced Slc9a6 ablation with tamoxifen and aged the mice to 14–16 months. Slc9a6 deficiency on the background of human ApoE4 reduced plaque deposition, as shown by Thioflavin S staining (Figure 7—figure supplement 3F, G) and 4G8 immunoreactivity (Figure 7C and D). The age-dependent increase in plaque load in ApoeAPOE4;AppNL-F mice was abolished or delayed when Slc9a6 was depleted after 2 months of age (Figure 8). AppNL-F mice expressing murine ApoE developed plaques at 12 months (Figure 7A and B), compared to ApoeAPOE4;AppNL-F which showed a similar number of plaques at 15–16 months (Figures 7C–D8, and Figure 7—figure supplement 3F-G). This delay of plaque deposition caused by the presence of human ApoE4 as opposed to murine ApoE is consistent with earlier findings by the Holtzman group (Liao et al., 2015). Importantly, NHE6/Slc9a6 ablation induced at 2 months showed a comparable reduction of plaque load as germline NHE6/Slc9a6 depletion. We conclude that plaque deposition is modulated by the presence of NHE6 postnatally and is not affected by NHE6 activity during development.

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).

NHE6/Slc9a6 deficiency does not affect cortical thickness and hippocampus size in AppNL-F;ApoeAPOE4 cortices

Xu et al., 2017, reported reduced cortex thickness and hippocampus volume in Slc9a6- mice, which we were able to reproduce in our germline Slc9a6- model (Figure 7—figure supplement 1). In addition, based on their findings Xu et al., 2017, concluded that the difference in brain size was a combined result of both neurodevelopmental and neurodegenerative effects caused by NHE6/Slc9a6 deficiency. Since both germline deficiency and adult-onset deficiency of NHE6 causes massive Purkinje cell loss in the cerebellum (Figure 3C–E), we next investigated the effect of conditional NHE6/Slc9a6 loss on hippocampal and cortical neuronal loss in our AppNL-F;ApoeAPOE4 model (Figure 7E–I). We measured brain size, cortical thickness, hippocampal area, and CA1 thickness. In contrast to germline Slc9a6- mice (Figure 7—figure supplement 1), none of the analyzed parameters differed significantly between Slc9a6fl;CAG-CreERT2 and controls (Figure 7E–I), and we specifically did not detect any reduction in brain size compared to Slc9a6fl;AppNL-F;ApoeAPOE4 littermate controls. This is consistent with the undergrowth model proposed by Xu et al., 2017, as we induced conditional disruption of NHE6/Slc9a6 in the adult after postnatal brain growth was completed.

NHE6 deficiency increases Iba1 and GFAP expression in the brain

Neuroprotective astrocytes and microglia have been described to reduce Aβ deposition in early stages of AD (Sarlus and Heneka, 2017). It has been reported that NHE6 deficiency leads to increased glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule (Iba1) immunoreactivity in different brain regions (Xu et al., 2017). To validate if plaque load correlated with Iba1 and/or GFAP immunoreactivity, we performed DAB immunostaining for both marker proteins. We found that Iba1 and GFAP immunoreactivity is increased in the white matter and to a lesser extent in the cortex of AppNL-F;Slc9a6- (Figure 9—figure supplement 1A, C, D, C, and D) and AppNL-F;ApoeAPOE4;Slc9a6fl;CAG-CreERT2 (Figure 9—figure supplement 1B, E, F) mice compared to their littermate controls. There was a non-significant trend toward increased immunoreactivity for both markers in the hippocampus of the Slc9a6- group. Taken together, these data are consistent with the findings of the Morrow group in germline Slc9a6- mice (Xu et al., 2017). Neurodegeneration can be a trigger for glial activation (Yanuck, 2019). However, our data on glial activation and cerebral volume in cKO mice, where NHE6/Slc9a6 was disrupted postnatally, suggest that neurodegeneration induced by NHE6/Slc9a6 deficiency is unlikely to be the trigger for the glial activation we observe.

Microglia and astrocytes surround plaques in both slc9a6- and control AppNL-F mice

Conditional and germline Slc9a6 deficient AppNL-F mice have a reduced number of plaques in the brain and an increase of Iba1 and GFAP labeled glia. Others have shown that Aβ plaques levels are reduced with increased plaque-associated microglia detected with a co-stain for Iba1 and Aβ (Parhizkar et al., 2019; Zhong et al., 2019). In order to investigate the contribution of microglia and astrocytes to the observed plaque reduction, we analyzed brain slices by co-staining for Aβ using the 6E10 antibody (Figure 9 and Figure 9—figure supplement 2). Whereas the total amount of plaques labeled by 6E10 was reduced in Slc9a6- as compared to control mice (Figure 9C), the amount of microglia and astrocytes surrounding plaques did not differ between genotypes (Figure 9D and G). In addition, there was no difference between genotypes in the amount of microglia co-labeled with 6E10 (Figure 9E) or the intensity of immunoreactivity for 6E10 within microglia (Figure 9F).

Figure 9 with 2 supplements see all
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).

Discussion

The prevalence of AD is increasing with life expectancy in all human populations. ApoE4 is the most important genetic risk factor. This makes it of paramount importance to understand the underlying mechanisms by which ApoE4 contributes to the pathology of the disease in order to devise effective targeted therapies that can be deployed on a global scale. Only small molecule drug therapies or, alternatively, immunization approaches can satisfy this requirement. Biologics, including monoclonal antibodies and potential viral gene therapy approaches, are unlikely to be sufficiently scalable. We have previously reported a novel small molecule intervention that has the potential to neutralize ApoE4 risk (Xian et al., 2018) through prevention of the ApoE4-induced endosomal trafficking delay of synaptic receptors by the early endosomal sorting machinery. The mechanistic basis of this conceptually novel intervention is the acidification of the early endosomal compartment through inhibition of NHE6. Remarkably and unexpectedly, loss of NHE6/Slc9a6 effectively reduced Aβ accumulation even in the absence of ApoE4, suggesting that hyperacidification of EEs prevents amyloid plaque formation independently of ApoE4. NHE6/Slc9a6 suppression or inhibition may thus be a universal approach to prevent amyloid buildup in the brain, irrespective of ApoE genotype. Our previous (Xian et al., 2018) and current studies thus suggest a novel mechanism to prevent ApoE4-risk for AD and delay plaque formation. In addition, genome-wide association studies (GWAS) in conjunction with studies on cell culture and mouse models of AD show that various AD risk factors enhance endolysosomal dysfunction (Knopman et al., 2021; Small et al., 2017; Verheijen and Sleegers, 2018), which potentially could be corrected by NHE6 inhibition.

Upon endocytosis, endosomes undergo gradual acidification controlled by vATPases which actively pump protons into the vesicular lumen, and by the Na+/H+ exchanger NHE6 which functions as a regulatable proton leak channel. NHE6 depletion acidifies early and recycling endosomes (Lucien et al., 2017; Ohgaki et al., 2010; Ouyang et al., 2013; Xinhan et al., 2011). pH is an important regulator of the endolysosomal sorting machinery in which vesicles undergo multiple rounds of fusion. EEs undergo fusion and fission events in close proximity to the cell membrane. Recycling endosomes originate from EEs while they undergo early-to-late endosomal maturation. In contrast to late endosomes, recycling endosomes do not undergo further acidification (Jovic et al., 2010; Schmid, 2017). The pH of EEs and recycling endosomes is ∼6.4–6.5 (Casey et al., 2010). This is normally sufficient to induce ligand receptor dissociation and enable cargo sorting. Our data, however, suggest that ApoE4 dramatically delays this fast recycling step in neurons, where ApoE, Apoer2, and glutamate receptors co-traffic through fast recycling compartments upon Reelin stimulation (Xian et al., 2018). We have proposed that ApoE4 impairs vesicle recycling due to isoelectric precipitation and structural unfolding at the physiological pH of the EE environment. This delays the dissociation of ApoE4 from its receptors, which in turn prolongs the entry of ApoE4 – along with Apoer2 and glutamate receptors in the same vesicle – into the recycling pathway (illustrated in Figure 1). We conclude that ApoE4 net charge affects its endosomal trafficking. This is further supported by recent findings by Arboleda-Velasquez and colleagues (Arboleda-Velasquez et al., 2019), who reported the presence of the ‘Christchurch’ R136S mutation in an E3/E3 PS1 mutation carrier without dementia. By neutralizing the positive charge of Arg136 in ApoE3, the IEP of this ApoE3 isoform is predicted to match that of ApoE2, which is protective against AD (Corder et al., 1994). ApoE2 homozygous carriers have an exceptionally low likelihood of developing AD (Reiman et al., 2020). Moreover, the Christchurch mutation is located within the heparin-binding domain of ApoE, which reduces its affinity for cell surface heparan sulfate proteoglycans. That in turn would result in decreased uptake and thus depletion of ApoE in EEs. The net effect would be unimpeded trafficking of EE vesicles through the fast recycling compartment.

In dendritic spines, NHE6 co-localizes with markers of early and recycling endosomes and with the glutamate receptor subunit GluA1. In the hippocampus, NHE6 is highly expressed in the pyramidal cells of the CA and the granule cells of the dentate gyrus (Strømme et al., 2011). Apoer2 is present at the postsynaptic density of CA1 neurons (Beffert et al., 2005). During LTP induction, translocation of NHE6-containing vesicles to dendritic spine heads is enhanced (Deane et al., 2013) and glutamate receptors are recruited to the synaptic surface through fast recycling (Fernández-Monreal et al., 2016). Our findings are consistent with a model where NHE6 serves as a pH regulator of Reelin-controlled fast recycling endosomes containing Apoer2 and glutamate receptors. This mechanism possibly translates to other cell types and other ApoE receptors.

We previously showed that ApoE4 impairs Reelin-mediated receptor recruitment to the neuronal surface and this can be reversed by functionally disabling NHE6 in primary neurons, which results in the increased acidification of EEs to a level sufficiently different from the IEP of ApoE4, which then allows its efficient dissociation from its receptors. Conditional Slc9a6 deletion accordingly alleviates the ApoE4-mediated resistance to Reelin-enhanced synaptic plasticity in hippocampal field recordings (Gao et al., 2019; Xian et al., 2018).

Aβ and tau, forming amyloid plaques and neurofibrillary tangles, are the defining features of AD pathology. As of today, it remains controversial how ApoE isoforms interfere with Aβ and tau pathology. ApoE, which is primarily expressed by astrocytes, is the major lipid transporter in the brain and in an isoform-dependent manner affects inflammatory, endolysosomal, and lipid-metabolic pathways (Gao et al., 2018; Minett et al., 2016; Van Acker et al., 2019; Xian et al., 2018). Most risk factors identified by GWAS, including but not limited to APOE, ABCA7, CLU, BIN1, TREM2, SORL1, PICALM, CR1, are members of one or more of these pathways (Kunkle et al., 2019). In recent years, endosomal dysfunction has increasingly gained acceptance as a causal mechanism for late-onset AD. Our findings now provide a mechanistic explanation how ApoE4 impairs endolysosomal trafficking and recycling, by interfering with vesicular sorting and maturation at a crucial bottleneck juncture of the endosomal trafficking machinery. This has far-reaching consequences for neuronal function, synaptic plasticity, and tau phosphorylation (Brich et al., 2003; Cataldo et al., 2000; Chen et al., 2005; Chen et al., 2010; Nuriel et al., 2017; Pensalfini et al., 2020). More specifically, ApoE4 causes abnormalities of Rab5-positive endosomes (Nuriel et al., 2017). Intriguingly, over-activation of the small guanosine triphosphatase (GTPase) Rab5, recapitulates neurodegenerative features of AD (Pensalfini et al., 2020).

ApoE4 alters APP processing and Aβ degradation (reviewed in Benilova et al., 2012; Haass et al., 2012; Huynh et al., 2017; Lane-Donovan and Herz, 2017; Pohlkamp et al., 2017; Yamazaki et al., 2019), and the ability of Reelin to protect the synapse from Aβ toxicity is impaired by ApoE4 (Durakoglugil et al., 2009). Aβ oligomerization followed by plaque formation is one hallmark of AD. NHE6 controls endosomal pH, which can affect BACE1 activity, one of the two enzymes required to process APP to release the Aβ peptide. Prasad and Rao, 2015, reported that overexpression of NHE6 and full-length APP in HEK293 cells, rather than its inhibition or knockdown, reduced Aβ generation, which conflicts with our findings in primary cortical neurons (Figure 6). Although the cause of this discrepancy remains currently unresolved, it is possible that it is the result of the two fundamentally different experimental systems that were used in the respective studies, that is, overexpression in immortalized kidney cells on one hand and primary cortical neuronal cultures on the other. Using a humanized AppNL-F knockin mouse model, we show that NHE6/Slc9a6 deficiency in 1-year-old animals reduces plaque deposition by ∼80%. In AppNL-F mice, plaques can be identified as early as 9 months of age. Whereas plaque deposition only increases by less than twofold between 9 and 12 months of age, it increases tenfold between 12 and 18 months (Saito et al., 2014). Importantly, the reduction in plaque deposition by Slc9a6 deficiency persists from early (12 months) to later stages (18 months) of AD, as Slc9a6-deficient AppNL-F animals aged 18 months had a reduction in insoluble Aβ by approximately 71%. NHE6/Slc9a6 depletion in AppNL-F;ApoeAPOE4 mice showed a comparable reduction in plaque load (Figure 7 and Figure 7—figure supplement 3). Our data are consistent with previous findings by the Holtzman group (Fagan et al., 2002) that showed that mouse ApoE promotes plaque deposition more potently than human ApoE4.

Prevention of plaque formation in our Slc9a6-deficient model was likely caused by increased microglial activation and plaque phagocytosis (Figure 7, Figure 7—figure supplement 3, 8,9Figure 8, Figure 9, Figure 9—figure supplement 1, and Figure 9—figure supplement 2). In the brains of AD patients and APP-overexpressing mice, plaques are surrounded by reactive microglia and astrocytes (Meyer-Luehmann et al., 2008; Serrano-Pozo et al., 2013), but the pathological significance of this is incompletely understood. Beneficial or detrimental roles of reactive microglia and astrocytes in the degradation of Aβ have been reported, depending on the activation state of these cells (Meyer-Luehmann et al., 2008; Ziegler-Waldkirch and Meyer-Luehmann, 2018). We observed an increase in reactive microglia and astrocytes resulting from NHE6/Slc9a6 depletion, which correlated with reduced plaque deposition in AppNL-F mice, irrespective of the presence of either murine ApoE or human ApoE4. As murine ApoE exacerbates plaque deposition even more than ApoE4, the comparable plaque reduction in Slc9a6-deficient mice with murine Apoe or ApoeAPOE4 might be the result of a maximally accelerated early endosomal maturation and cargo transport in the absence of NHE6. When compared to control AppNL-F mice, Slc9a6-deficient AppNL-F mice show an increase in Iba1 (microglia) and GFAP (astrocytes) immunoreactivity, but reduced Aβ immunoreactivity. Surprisingly, the intensity of GFAP and Iba1 in plaque areas was comparable between the groups. Moreover, even though Aβ is reduced and Iba1 is increased in the Slc9a6-, the proportion of microglial structures containing Aβ (6E10 antibody) was comparable between Slc9a6-deficient and control AppNL-F mice, as was the intensity signal for 6E10. This suggests that microglia in the Slc9a6- may be more efficient in taking up and degrading Aβ. Whether the reduction in plaques is due to the presence of an increased number of microglia and astrocytes that actively phagocytose nascent plaques, or whether endosomal acidification in microglia and astrocytes improves their ability to degrade or export Aβ from the brain remains to be determined. It is tempting to speculate that the mechanism that leads to reduced plaque load in Slc9a6-deficient AppNL-F mice may involve an increased catabolic rate (Shi et al., 2021), brought about by the accelerated acidification and vesicular trafficking of EEs.

It is also possible that Slc9a6 deficiency alters the efficiency of astrocytes to lipidate ApoE. In a mouse model, improved ApoE lipidation by the overexpression of ATP-binding cassette transporter family member A1 (ABCA1) decreased plaque deposition (Wahrle et al., 2005; Wahrle et al., 2008; Wahrle et al., 2004). During HDL assembly, ABCA1 shuttles between EE and the plasma membrane, a process also referred to as retroendocytosis (Ouimet et al., 2019). Moreover, membrane trafficking of ABCA1 is altered by ApoE in an isoform-dependent fashion (Rawat et al., 2019). The AppNL-F mouse model used in our study develops plaques at 12 months (Saito et al., 2014) in the presence of murine ApoE. However, the onset of plaque deposition in human ApoeAPOE4 mice was delayed by ∼3 months. The effect of germline Slc9a6 deficiency and conditional Slc9a6 deficiency induced at 2 months had a comparable effect on plaque reduction in AppNL-F and AppNL-F;ApoeAPOE4 mice.

Slc9a6- and Slc9a6fl;CAG-CreERT2 both show progressive Purkinje cell loss in the cerebellum, indicating that NHE6 requirement is cell-autonomous and not developmentally determined. Slc9a6- and Slc9a6fl;CAG-CreERT2 show a comparable increase in immunoreactivity against Iba1 and GFAP. Increased glia reactivity can be a direct cell-autonomous effect of NHE6 loss or an indirect effect caused by NHE6 deficiency-related neurodegeneration. As germline Slc9a6- mice present with a reduction in cortical thickness and hippocampal volume caused by both neurodevelopmental and neurodegenerative effects (Xu et al., 2017; Figure 7—figure supplement 1), it is possible that neurodegeneration gives rise to glia activation. However, the tamoxifen-induced Slc9a6fl;CAG-CreERT2 mice do not present with a reduction in cortical or hippocampal volume (Figure 7), yet have comparable immunoreactivity for markers of glial activation. Our study supports a temporally distinct function of NHE6 in the adult brain where the cerebrum requires NHE6 for development, but not for postnatal neuronal survival, whereas Purkinje cells in the cerebellum do. A similar dual function for NHE6 has been described previously by Xu et al., 2017. Moreover, this further indicates that reduced plaque load is also not an effect of neuronal loss. Therefore, our two mouse models together suggest that the observed increased glial activation is not caused by neuronal cell loss, but rather is likely a direct cell-autonomous effect of NHE6 loss of function. It thus remains to be determined whether endosomal acidification in NHE6-deficient microglia alone is sufficient to induce Aβ degradation and plaque reduction.

NHE6 is ubiquitously expressed in all cells of the body, however, in the CNS it is highly expressed in neurons where abundant synaptic vesicles and neurotransmitter receptors recycle in synapses (Lee et al., 2020; Lee et al., 2021) and to a lesser extent in glial cells (Zhang et al., 2014). Neuronal NHE6 plays a direct role in both synaptic development and plasticity, potentially through BDNF/TrkB (Deane et al., 2013; Ouyang et al., 2013) and other pathways. The impact of NHE6 loss in microglia and astrocytes is still unknown. Global Slc9a6 deficiency causes glial activation in vivo, which could be mediated through either a primary cell-autonomous mechanism or a secondary mechanism induced by damaged neurons. In ApoE4-expressing astrocytes, others have shown that overexpression of NHE6 increases LRP1 on the surface, which correlated with an increase in Aβ uptake (Prasad and Rao, 2018). Microglia are the primary glial cells that degrade Aβ; thus, it will be imperative to determine how NHE6 levels alter Aβ degradation selectively in astrocytes and microglia, respectively.

Early endosomal pH balance in phagocytic cells plays an important role in viral and bacterial infection response. In phagocytic cells, the deacidification of endosomes using pharmacological inhibitors like chloroquine has been shown to reduce endosomal toll-like receptor (TLR) response (de Bouteiller et al., 2005; Fox, 2019; Kuznik et al., 2011; Wozniacka et al., 2006; Yang et al., 2016), suggesting that NHE6 depletion in microglia might conversely augment TLR response.

It would also be intriguing to test whether Slc9a6 deficiency increases cognitive performance in AD mouse models. In the current study we used AppNL-F and AppNL-F;ApoeAPOE4 mice as the most physiological currently available mouse models of AD that do not rely on excessive amyloid overproduction. These mice, however, do not show cognitive impairments in spatial learning tests like Morris water maze (Saito et al., 2014) (own unpublished observations). Other neurobehavioral phenotypes have been described for Slc9a6- mice, which also recapitulate symptoms in Christianson syndrome patients, for example, hyposensitivity to pain (Petitjean et al., 2020). Future studies on our novel tamoxifen-inducible Slc9a6fl;CAG-CreERT2 line will help to understand whether these symptoms are based on neurodevelopmental deficits caused by germline Slc9a6 deficiency or whether they can be reproduced by induced loss of NHE6 postnatally.

In conclusion, we have shown that both the endosomal trafficking defect induced by ApoE4 in neurons and increased plaque deposition irrespective of ApoE genotype can be corrected by inhibition or genetic deletion of NHE6/Slc9a6, a key regulator of early endosomal pH. Accelerated acidification of EEs abolishes the ApoE4-induced Reelin resistance and restores normal synaptic plasticity in ApoE4-targeted replacement mice. The first FDA-approved drug for AD treatment in 18 years is aducanumab (Sevigny et al., 2016), an antibody directed against Aβ, which clears amyloid from the brain. However, amyloid removal in individuals already afflicted with AD provides at best marginal benefits at this stage. Moreover, in excess of 1 billion people worldwide are ApoE4 carriers, making early treatment with a complex biologic such as aducanumab impractical on the global scale. Here, we have presented a potential alternative approach which should be adaptable to large-scale prevention treatment using blood-brain barrier penetrant NHE6-specific inhibitors. Taken together, our combined data suggest that endosomal acidification has considerable potential as a novel therapeutic approach for AD prevention and possibly also for the prevention of disease progression.

Materials and methods

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

Animals

All animal procedures were performed according to the approved guidelines (Animal Welfare Assurance Number D16-00296) for Institutional Animal Care and Use Committee (IACUC) at the University of Texas Southwestern Medical Center at Dallas.

The mouse lines Rosa-stop-tdTomato B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Madisen et al., 2010) (JAX #007909) and CAG-CreERT2 B6.Cg-Tg(CAG-cre/Esr1)5Amc/J mice (Hayashi and McMahon, 2002) (JAX #004682) were obtained from The Jackson Laboratories (Bar Harbor, ME). ApoE3 and ApoE4 targeted replacement mice (ApoeAPOE3, ApoeAPOE4) (Knouff et al., 1999; Sullivan et al., 1997) were kind gifts of Dr Nobuyo Maeda. AppSwe (Tg2576) were generated by Hsiao et al., 1996. The Meox-Cre B6.129S4-Meox2tm1(cre)Sor/J mice (JAX 003755) were provided by Drs M Tallquist and P Soriano (Tallquist and Soriano, 2000). Conditional NHE6 KO (Slc9a6fl;CAG-CreERT2) and germline NHE6 KO (Slc9a6-) mice were generated as described below. The human APP knockin line (AppNL-F) (Saito et al., 2014) has been described earlier. Pregnant female SD (Sprague Dawley) rats were obtained from Charles River (SC:400). Mice were group-housed in a standard 12 hr light/dark cycle and fed ad libitum standard mouse chow (Envigo, Teklad 2016 diet as standard and Teklad 2018 diet for breeding cages).

To generate Slc9a6-floxed (Slc9a6fl) mice, the first exon of NHE6 was flanked with loxP sites (Figure 2A). A loxP site was inserted 2 kb upstream of the first exon of the X-chromosomal NHE6 gene and a Neo-cassette (flanked with loxP and FRT sites) was inserted 1 kb downstream of the first exon. Insertion sites were chosen based on low conservation (mVISTA) between mammalian species (rat, human, mouse). To create the targeting vector for the Slc9a6fl mouse line, pJB1 (Braybrooke et al., 2000) was used as backbone. Murine C57Bl/6 J embryonic stem (ES) cell DNA was used as template to amplify the short arm of homology (SA; 0.88 kb of the first intron starting 1 kb 3’ downstream of exon 1), which was inserted between the Neo and HSVTK selection marker genes of pJB1 (BamHI and XhoI sites) to create an intermediate plasmid referred to as pJB1-NHE6SA. To create the intermediate plasmid pNHE6-LA for the long homology arm, a fragment spanning from 13 kb 5’ upstream to 1 kb 3’ downstream of the first Slc9a6 (NHE6) exon was integrated into pCR4-TOPO (Thermo Fisher) by using a bacterial artificial chromosome (RP23 364F14, Children’s Hospital Oakland Research Institute [CHORI]) and the GAP repair technique (Lee et al., 2001) (primers to amplify the upstream (US) and downstream (DS) homology boxes are listed in the Key resources table). In a parallel cloning step, a 2.4 kb XhoI-EcoRV Slc9a6 promoter region fragment spanning from 2.7 to 0.4 kp 5’ upstream of the Slc9a6 start-codon was modified with the 1st loxP site to generate pLoxP: three fragments (1) 0.7 kb 5’ loxP flanking Slc9a6-fragment, (2) 1.7 kb 3’ loxP flanking Slc9a6 fragment, and (3) 100 bp loxP oligo (primers/oligos for each fragment are listed in the Key resources table) were cloned into pCR4-TOPO. The 2.5 kb loxP-modified XhoI-EcoRV Slc9a6-promoter fragment of pLoxP was cloned into pNHE6-LA to create pNHE6-LA-LoxP. To obtain the final targeting construct pJB-NHE6-TV, the NotI-EagI fragment of pNHE6-LA-LoxP containing the long arm (11 kb 5’ upstream of the 1st LoxP) and the 1st loxP site was cloned into the NotI-site of pJB1-NHE6SA and checked for orientation (pJB1-NHE6 targeting vector). pJB-NHE6-TV was linearized with NotI and electroporated into C57Bl/6 J ES cells. Gene targeting-positive C57Bl/6 J ES cells (PCR screen) were injected into albino C57Bl/6 J blastocysts, resulting in chimeric mice. The chimeras were crossed to C57Bl/6 J mice, resulting in Slc9a6fl/+ females. Genotyping: The Slc9a6fl PCR amplifies a 250 bp of the wildtype and 270 bp of the floxed allele, primers are listed in the Key resources table.

To generate Slc9a6fl;CAG-CreERT2, Slc9a6fl/+ females were crossed to CAG-CreERT2 mice to obtain Slc9a6fl/fl;CAG-CreERT2 and Slc9a6y/fl;CAG-CreERT2 mice (Slc9a6fl;CAG-CreERT2) and CAG-CreERT2 negative control littermates (Slc9a6fl). Slc9a6fl;CAG-CreERT2 mice were backcrossed to ApoeAPOE3 or ApoeAPOE4 mice. Breeding pairs were set up in which only one parent was CAG-CreERT2 positive. The following genotypes were used for hippocampal field recordings: (1) ApoeAPOE3;SLC9a6y/fl (short:ApoeAPOE3), (2) ApoeAPOE3;Slc9a6y/fl;CreERT2 (short: ApoeAPOE3; Slc9a6fl;CAG-CreERT2), (3) ApoeAPOE4;Slc9a6y/fl (short: ApoeAPOE4), and (4) ApoeAPOE4;SLc9a6y/fl;CreERT2 (short: ApoeAPOE4;Slc9a6fl;CAG-CreERT2). The ApoeAPOE4;Slc9a6fl;CAG-CreERT2 line was further crossed with the AppNL-F line to generate ApoeAPOE4;Slc9a6fl;CAG-CreERT2;AppNL-F and ApoeAPOE4;Slc9a6fl;AppNL-F control littermates. To induce genetic depletion of NHE6, tamoxifen (120 mg/kg) was intraperitoneally injected at 6–8 weeks of age. Injections were applied for 5 consecutive days. Tamoxifen was dissolved in sunflower oil (Sigma, W530285) and 10% EtOH.

To generate the germline NHE6 KO line (Slc9a6-), heterozygous Slc9a6fl/+ females were crossed to Meox-Cre to yield germline mutant Slc9a6-/- females and Slc9a6y/- males (Slc9a6-). Genotyping: The Slc9a6fl PCR amplifies 250 bp of the wildtype, 270 bp of the floxed, and no fragment in the KO alleles. Recombination was verified with the NHE6-rec PCR, which amplifies 400 bp if recombination has occurred. PCR primers are listed in the Key resources table. Slc9a6- animals were further crossed with AppNL-F (Saito et al., 2014) mice. AppNL-F;Slc9a6- (AppNL-F/NL-F;Slc9a6y/- or AppNL-F/NL-F;Slc9a6-/-) and control (AppNL-F = AppNLNL-F/NL-F;Slc9a6y/+ or AppNL-F/NL-F;Slc9a6+/+) littermates were obtained by crossing Slc9a6+/-;AppNL-F/NL-F females with either Slc9a6y/+;AppNL-F/NL-F or Slc9a6y/-;AppNL-F/NL-F males.

DNA constructs, recombinant proteins, lentivirus production

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Lentiviral plasmids with shRNA directed against NHE6/Slc9a6 and the scrambled control have been described in Xian et al., 2018. Plasmids encoding ApoE3 and ApoE4 (Chen et al., 2010), and Reelin D’Arcangelo et al., 1997 have been described before. The lentiviral plasmid encoding the Vamp3-pHluorin2 fusion protein was cloned by inserting mouse Vamp3 cDNA, a linker and pHluorin2 (pME, Addgene #73794) (Stawicki et al., 2014) into pLVCMVfull (Xian et al., 2018). For Vamp3 the forward primer (5’- TTCAAGCTTCACCATGTCTACAGGTGTGCCTTCGGGGTC-3’) contains a Kozak sequence, the reverse primer encodes a KLSNSAVDGTAGPGSIAT linker (Nakamura et al., 2005) (5’ CATTGTCATCATCATCATCGTGTGGTGTGTCTCTAAGCTGAGCAACAGCGCCGTGGACGGCACCGCCGGCCCCGGCAGCATCGCCACCAAGCTTAAC-3'). The pHluorin2 primers were forward 5’-CCGGTCCCAAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTC-3’ and reverse 5’- GCCCTCTTCTAGAGAATTCACTTGTACAGCTCGTCCATGCCGTG-3’. The fragments were sequentially cloned into pcDNA3.1 and the fusion protein was then transferred with NheI and EcoRI into pLVCMVfull. Lentiviral plasmids psPAX2 and pMD2.g were a kind gift from Dr D Trono and obtained from Addgene.

Recombinant Reelin and ApoE were generated in HEK293 cells. Reelin was purified as described before (Weeber et al., 2002). ApoE-conditioned medium was collected from HEK293 cell cultures transiently transfected with pcDNA3.1-ApoE constructs or empty control vector (pcDNA3.1-Zeo). ApoE concentration was measured as described before (Xian et al., 2018).

For lentivirus production HEK 293 T cells were co-transfected with psPAX2, pMD2.g, and the individual shRNA encoding transfer or Vamp3-pHluorin2 constructs. Media was replaced after 12–16 hr. Viral particle containing media was collected after centrifuging cellular debris. The viral particles were 10× concentrated by ultra-centrifugation and resuspended in Neurobasal medium. To infect neurons on DIV7 1/10th of the culture medium was replaced by concentrated virus. After 24 hr, the virus was removed.

Histochemistry

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Mice were euthanized with isoflurane and perfused with PBS followed by 4% paraformaldehyde (PFA) in PBS. Brains were removed and post-fixed for 24 hr in 4 % PFA. Fresh fixed brains were immobilized in 5 % agarose in PBS and 50 μm thick sections were sliced on a vibratome (Leica, Wetzlar, Germany). Vibratome slices of Rosa26floxedStop-tdTomato;CAGCreERT2 mice, with or without tamoxifen injection at 8 weeks of age were mounted with Antifade Mounting medium containing DAPI (Vectashield). For immunofluorescence, vibratome slices were labeled for Calbindin after permeabilization with 0.3 % Triton X in PBS and blocking for 1 hr in blocking buffer (10 % normal goat serum, 3 % BSA, and 0.3 % Triton X in PBS). The primary antibody mouse anti-Calbindin (Swant CB300) was diluted in blocking buffer (1:1000) and added to the slices for 24–48 hr at 4°C. Slices were subsequently washed 4 × 15 min with PBS containing 0.3 % Triton X. Slices were incubated with anti-mouse IgG coupled to Alexa594 (1:500 in blocking buffer) for 2 hr at room temperature. After washing, slices were mounted with Antifade Mounting medium with DAPI (Vectashield). Images were taken with an Axioplan two microscope (Zeiss).

For immunohistochemistry staining, PFA-fixed brains were block-sectioned into coronal slabs, paraffin-embedded, and serially sectioned on a rotating microtome (Leica) at 5 μm. Deparaffinized sections were stained with Thioflavin S as described before (Guntern et al., 1992). Briefly, deparaffinized slices were oxidized in 0.25 % KMnO4 for 20 min. After washing with water, slices were bleached with 1 % K2S2O5 / C2H2O4 for 2 min, washed in water, and treated with 2 % NaOH/H2O2 for 20 min. After washing with water, slices were acidified in 0.25 % CH3COOH for 1 min, washed with water and equilibrated in 50 % EtOH for 2 min, and stained in Thioflavin S solution for 7 min. Reaction was stopped by washing in 50 % EtOH. Slices were dehydrated with 95 % EtOH, followed by 100 % EtOH and xylene. Slices were mounted with Cytoseal 60 (Thermo Scientific). Deparaffinized sections were labeled using antibodies raised against GFAP (Abcam AB5804, Rabbit, 1:2000), Iba1 (Wako 019–19741, Rabbit, 1:1000), and Aβ (4G8, Covance or 6E10, Biolegend, both mouse, 1:1000). Briefly, 5 μm sections were deparaffinized, subjected to microwave antigen retrieval (citrate buffer, pH 6.0), permeabilized with 0.3 % (vol/vol) Triton X, endogenous peroxidases activity was quenched for diaminobenzidine (DAB) staining. Slices were blocked with goat serum (2.5%) prior to overnight incubation with primary antibodies at 4°C. Primary antibodies were detected by either fluorescent secondary antibodies (goat-anti-mouse Alexa594, goat-anti-rabbit Alexa488) or sequential incubation with biotinylated secondary antisera and streptavidin-peroxidase for DAB staining. DAB chromagen was used to detect the immunoperoxidase signal (Sinclair et al., 1981) (Vector; anti-mouse and anti-rabbit IgG kits). Fluorescence-labeled slices were counterstained with DAPI (mounting media with DAPI, Vectashield). Standard protocols were utilized for staining of paraffin sections with H&E (Leica) (Fischer et al., 2008). Microscopy was performed on a high-throughput microscope (NanoZoomer 2.0-HT, Hamamatsu) for DAB-stained tissue or with an Axioplan two microscope (Zeiss) for immunofluorescence analysis. The analysis of DAB-labeled antibodies was performed after exporting the images with NDP.view2 software with ImageJ. For Thioflavin S staining and Aβ labeling plaques were quantified by categorizing them as small, medium, and large (Thioflavin S) or dense and diffuse (4G8) as depicted in Figure 7—figure supplement 2. Co-localization analysis of microglia (Iba1) and astrocytes (GFAP) with plaques (6E10) was performed with ImageJ. A blind observer selected the area of plaques with circles of 20, 40, or 80 µm diameter and analyzed the intensity of 6E10 and Iba1 or GFAP by using the ImageJ plugin RGB_measure. 6E10-positive microglia (Iba1) were quantified by a blind observer by first identifying microglia structures/cells in the green channel (Iba1), and then analyzing the proportion of 6E10-positive structures and signal intensity (red channel).

Primary cortical neuronal cultures

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Primary cortical neuronal cultures were prepared from rat (SD, Charles River) or various mouse lines (wildtype, Slc9a6-, AppSwe) (E18) as described previously (Chen et al., 2005). Neurons were cultured in poly-D-lysine coated six-well plates (1 million/9 cm2) or on coverslips (30,000 neurons/1.1 cm2) in presence of Neurobasal medium supplemented with B27, glutamine, and penicillin-streptomycin at 37°C and 5% CO2. At indicated days in vitro (DIV) primary neurons were used for experiments.

Mouse embryonic fibroblasts

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Fibroblasts were isolated from Slc9a6- and wildtype littermate control embryos (E13.5). After removing the head and the liver, the tissue was trypsinized (0.05% trypsin-EDTA) at 4°C overnight, followed by 30 min at 37°C. Suspended cells were cultivated in DMEM high glucose with 15% FCS, 2 mM L-glutamine, and Pen/Strep. Fibroblasts were serially passaged until proliferation slowed down. Immortalization was achieved by keeping fibroblasts in culture under high confluency until they overcame their growth crisis.

pH measurements

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Mouse embryonic fibroblasts derived from either Slc9a6- or wildtype littermate embryos were infected with Vamp3-pHluorin2 lentivirus; 24–48 hr post-infection, vesicular pH was measured on a Zeiss LSM880 Airyscan confocal microscope as described in Ma et al., 2017. For the standard calibration curve, cells were washed and incubated with pH standard curve buffer (125 mM KCl, 25 mM NaCl, 10 μM monensin, 25 mM HEPES for pH >7.0 or 25 mM MES for pH <7.0; pH adjusted with NaOH and HCl) and imaged in 5% CO2 at 37°C. For vesicular pH measurements, cells were washed and imaged with pH standard curve at pH 7 without monensin. Samples were excited at 405 and 488 nm with an emission of 510 nm. For quantification, six fields of view were imaged for Slc9a6+ and four fields of view for Slc9a6- fibroblast. Between 4 and 10 pHluorin-positive vesicles were measured per field of view which resulted in n = 32 wildtype vesicle and n = 28 Slc9a6- vesicles. The same settings were used for every image, and images were analyzed using ImageJ software. The intensity of excitations with 405 and 488 nm was measured, individual vesicles were marked as regions of interest, and the 405/488 ratio was calculated and plotted against pH for the standard curve.

Biochemistry

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To analyze receptor recycling, cell surface biotinylation was performed. At DIV10–14, primary neurons were pre-treated for 30 min with ApoE-conditioned medium (5 μg/ml) and incubated with Reelin (2 μg/ml) for an additional 30 min (see timeline in Figure 4A). After treatment, cells were washed with cold PBS and incubated in PBS containing sulfo-NHS-SS-biotin for 30 min at 4°C. Subsequently excess reagent was quenched by rinsing the neurons with cold PBS containing 100 mM glycine. Neurons were lysed in 160 µl/9 cm2 lysis buffer (PBS with 0.1% SDS, 1% Triton X-100, and protease inhibitors) at 4°C for 20 min. Cell debris were pelleted at 14,000 rpm for 10 min at 4°C. The protein concentration was measured using the Bradford Protein Assay (Bio-Rad). One hundred µg of total proteins were incubated with 50 µl of NeutrAvidin agarose at 4°C for 1 hr. Agarose beads were washed three times using washing buffer (500 mM NaCl; 15 mM Tris-HCl, pH 8.0; 0.5% Triton X-100), biotinylated surface proteins were eluted from agarose beads by boiling in 2× SDS sample loading buffer and loaded on an SDS-PAGE gel for Western blot analysis. GST-control and GST-RAP (50 μg/ml) pre-treatment of neurons was performed for 1 hr.

To analyze BACE1 activity, β-CTF was detected by immunoblotting. BACE1 activity was examined after pharmacological NHE inhibition or genetic Slc9a6 knockdown in primary neurons of AppSwe mice. For NHE inhibition DIV10 neurons were treated with 5 µg/ml ApoE4, 3 µM EMD87580 (Merck), and/or 1 µM γ-secretase inhibitor L-685458 (Merck) for 5 hr. For knockdown of NHE6/Slc9a6 DIV7 neurons of AppSwe mice were infected with lentivirus encoding shRNA against NHE6 or a scrambled control shRNA. On DIV13 neurons were treated with γ-secretase inhibitor for 12 hr. Proteins were extracted for Western blot analysis: Cells were washed three times with cold PBS and lysed for 20 min on ice in RIPA buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Nonidet P-40; phosphatase and proteinase inhibitors). Cell debris were pelleted at 14,000 rpm for 10 min at 4°C. Protein concentration was measured using the Bradford Protein Assay (Bio-Rad). After adding 4× SDS loading buffer (0.1 M Tris-HCl, pH 6.8, 2% SDS, 5% β-mercaptoethanol, 10% glycerol, and 0.05% bromophenol blue), samples were denatured at 80°C for 10 min. Proteins were separated via SDS-PAGE and transferred to a nitrocellulose membrane for Western blotting using different antibodies listed in the Key resources table.

Brain tissue was dissected and prepared for immunoblotting as follows: After removal, brains were placed in ice-cold PBS containing proteinase inhibitors. Anatomical sectioning was performed under a microscope. The hippocampus was dissected out and transversal slices were further separated into cornu ammonis (CA) 1, CA3, and dentate gyrus. Respective pieces of the same anatomical regions of one brain were pooled, shock-frozen in liquid nitrogen, and stored at –80C. Frozen tissue was homogenized in 10× volume of RIPA buffer and incubated on ice for 30 min. Cell debris were pelleted for 10 min with 14,000 rpm at 4°C. After adding 4× SDS loading buffer, the samples were denatured at 80°C for 10 min and used for immunoblotting.

To measure soluble and insoluble Aβ species, a sequential homogenization procedure was employed. After removal of the brains from PBS perfused mice, cortical tissue was dissected and flash-frozen. Frozen cortical tissue was homogenized in TBS supplemented with phosphatase and proteinase inhibitors at 100 mg protein/ml using a glass dounce homogenizer. Crude lysate was centrifuged at 800 × g for 5 min at 4°C. The supernatant was further centrifuged at 100,000 × g for 30 min at 4°C and collected as TBS-soluble lysate (Aβ-soluble). The TBS pellet was further homogenized in 1% Triton-TBS containing phosphatase and proteinase inhibitors, centrifuged at 100,000 × g for 30 min at 4°C and collected as 1% Triton-soluble lysate. The Triton-pellet was incubated with 5 M guanidine-HCl rotating at RT for 1 hr. Guanidine-soluble lysate (Aβ-insoluble) was collected after centrifugation at 21,000 × g for 15 min at 4°C. The guanidine-pellet was further solubilized in 1/20th volume with 70% formic acid (Aβ-insoluble) and centrifuged at 21,000 × g for 15 min at 4°C. Soluble and insoluble Aβ levels were measured in duplicates using a commercial Aβ42 ELISA kit (Thermo Fisher, KHB3441) following the manufacturer’s instructions.

Extracellular field recordings

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Hippocampal slices were prepared from 3-month-old mice (tamoxifen-injected at 8 weeks). Slices of mice were obtained from four different genotypes; Slc9a6fl;CAG-CreERT2 mice or Slc9a6fl mice with ApoeAPOE3 or ApoeAPOE4. The brains were quickly removed and placed in ice-cold high sucrose cutting solution (in mM: 110 sucrose, 60 NaCl, 3 KCl, 1.25 NaH2PO4, 28 NaHCO3, 0.5 CaCl2, 5 glucose, 0.6 ascorbic acid, 7 MgSO4), bubbled with a gas mixture of 95% O2 and 5% CO2 for oxygenation. 350 µm transverse sections were cut using a vibratome (Leica). Slices were transferred into an incubation chamber containing 50% artificial cerebrospinal fluid (aCSF, in mM: 124 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 D-glucose, 2 CaCl2, 1 MgSO4) and 50% sucrose cutting solution, oxygenated with 95% O2/5% CO2. Slices were transferred into an oxygenated interface chamber and perfused with aCSF with or without Reelin (2 μg/ml). The stimulating electrode was placed on the Schaffer-collateral of the CA1-pyramidal neurons and the recording electrode on the dendrites of the CA3-pyramidal neurons. Once baseline was stably recorded for 20 min, theta burst was applied, and traces collected for an hour. For stimulation concentric bipolar electrodes (FHC, catalog no CBBRC75) were placed into the stratum radiatum. Stimulus intensity was set at 40–60% of maximum response and delivered at 33 mHz through an Isolated Pulse Stimulator (A-M Systems, Model 2100). A custom written program in LabView 7.0 was used for recording and analysis of LTP experiments. A theta burst (TBS; train of four pulses at 100 Hz repeated 10 times with 200 ms intervals; repeated five times at 10 s intervals) was used as a conditioning stimulus.

Statistical methods

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Data were expressed as the mean ± SEM and evaluated using two-tailed Student’s t-test for two groups with one variable tested and equal variances, one-way analysis of variance (ANOVA) with Dunnett’s post hoc or Tukey’s post hoc for multiple groups with only variable tested, or two-way ANOVA with Sidak’s post hoc for plaque quantification (two independent variables of NHE6 genotype and plaque classification). The differences were considered to be significant at p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001). Software used for data analysis was ImageJ (NIH), LabView7.0 (National Instruments), Odyssey Imaging Systems (Li-Cor), Prism7.0 (GraphPad Software).

Data availability

All relevant data are included in the manuscript.

References

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Decision letter

  1. Jeannie Chin
    Reviewing Editor; Baylor College of Medicine, United States
  2. Jonathan A Cooper
    Senior Editor; Fred Hutchinson Cancer Research Center, United States
  3. Eric Morrow
    Reviewer; Brown University

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This paper is of interest to a broad range of neuroscientists, particularly those interested in ApoE biology and Alzheimer's disease (AD), as it reveals a novel mechanism that counteracts AD-linked amyloid plaque burden and synapse dysfunction in mice. Overall, the methodology is sound, sophisticated, and employs animal models that more closely mimic human diseases, and the results are compelling.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Endosomal Acidification by NHE6-depletion Corrects ApoE4-mediated Synaptic Impairments and Reduces Amyloid Plaque Load" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Eric Morrow (Reviewer #2).

We are sorry to say that, after consultation with the reviewers, we have decided that your work will not be considered further at this time for publication by eLife.

We would like to emphasize that all 3 reviewers found considerable strengths in your work, particularly the marked reductions in plaques in mice in which NHE6 was ablated or reduced. However, the lack of evidence that microglia and/or astrocytes are activated, take up Abeta, and mediate the plaque clearance in your studies leaves room for alternative explanations that were not addressed. Therefore, the data as they stand now do not strongly confirm the proposed mechanistic hypothesis put forward in your manuscript. This and other concerns listed below in the Reviewers' comments may take substantial time to satisfactorily address, which was the basis for our decision. If you decide to perform additional studies that address these concerns, we encourage you to consider submitting your thoroughly revised manuscript again to eLife.

Reviewer #1:

In this study, the authors confirm and extend their previous work demonstrating that ApoE4, a major risk for Alzheimer's disease, impairs endocytic recycling of membrane receptors, leading to synaptic dysfunction. Previously, the authors demonstrated in vitro that upon binding to ApoER2 at the plasma membrane and internalization, ApoE4 along with ApoER2 and glutamate receptors become trapped in the early endosome due to the similarity between the isoelectric point of ApoE4 and the pH of early endosomes. Enhancing acidification by inhibiting NHE6, a proton leak channel in the early endosome, restored vesicle recycling and improved synaptic plasticity in AD extract-treated hippocampal slices from ApoE4-KI mice. In the current study, the authors create and use novel NHE6 germline knockout and conditional knockout mouse lines to reduce NHE6 expression and enhance acidification of early endosomes. They confirm their previous findings and also extend their work by crossing NHE6 KO or cKO mice to a knockin, humanized APP mouse that expresses mutant human amyloid precursor protein under control of the endogenous APP promoter, alone or crossed with ApoE4-KI mice. In both cases, reduction of NHE6 resulted in increased Iba1-expressing microglia and GFAP-expressing astrocytes as well as a reduction in plaques. Together these findings highlight the importance of ApoE4's detrimental effect on endosomal recycling in vivo, with consequences for accumulation of AD-related pathology. While the studies presented are well-done and robust, some mechanistic links are missing, which make it difficult to fully support the conclusions drawn.

Major strengths of this paper include:

1. The creation of novel germline KO and conditional KO NHE6 mice allow for a number of in vivo investigations that would be difficult to complete otherwise. These mice represent a valuable resource to the field.

2. Use of the NHE6-KO and cKO mice to confirm the previous findings (that used pharmacological inhibition of NHE6) that enhancing endosomal acidification ameliorates ApoE4-induced deficits in vesicle cycling and synaptic plasticity in vivo. In addition, the finding that NHE6 ablation in APP and APP/ApoE4KI mice robustly reduced plaque accumulation is striking.

3. The demonstration that BACE-mediated production of APP CTFs is unaltered by NHE6 ablation supports the conclusion that the reduction in plaques is unlikely due to reduction in Abeta generation, but more likely due to clearance of Abeta.

Weaknesses of this paper include:

1. The authors conclude that reducing NHE6 clears plaques by activating resident microglia, shifting them from a dormant state to a damage-associated activated state that phagocytoses Abeta plaques. However, there is no data presented to demonstrate this. In a supplemental figure, the authors show there are more Iba1-expressing microglia and GFAP-expressing astroctyes in APP mice and in APP/ApoE4KI mice in which NHE6 has been ablated, but this does not prove that this is the mechanism by which plaques are cleared.

2. The mechanisms underlying the increase in Iba1 and GFAP are not clear. The authors cite a previous paper from another group that demonstrated in their own NHE6 KO mice, there was an increase in GFAP and in activated microglia expressing CD68, which may relate to the cell loss in hippocampus and other brain regions documented in those mice. However, in the current study, the authors indicate that in their NHE6 KO lines, there is no overt cell loss. It is therefore unclear how reductions in NHE6 expression lead to microglial/astrocyte activation. This is an important point to work out, since the authors conclude that it is microglial activation that is responsible for the reduction in Abeta plaques.

3. What might be some of the underlying explanations be for the differences between the published NHE6-KO mice, which has fairly widespread cell loss, and the current KO mice generated in this paper, which did not exhibit noticeable cell loss in brain regions other than the cerebellum?

4. There are a number of mechanistic links that have not been worked out, as indicated above. Until these links are identified and characterized, a number of the conclusions drawn by the authors are not yet supported.

Specific suggestions for authors:

1.To better assess whether the Iba1-expressing microglia are truly activated, CD68 should be stained. It would also be extremely compelling to stain for Abeta and demonstrate increased Abeta inside of microglia.

2. In order to conclude that NHE6 ablation clears plaques BY activating microglia, the authors should deplete microglia and then see whether there is still an effect on plaque load. If there is, that would firmly support their hypothesis; but if eliminating microglia has no effect on plaques, it would suggest that there are other mechanisms at play.

3. Careful assessment of cell viability/death in the NHE6 mice (and related crosses) should be done. It is puzzling why the NHE6 KO lines here would show such differences in level of cell loss, relative to the study by Xu et al. It is an important point though, because (1) it could help provide a mechanism by which microglia get activated. (2) it is necessary to fully appreciate the LTP studies – the Xu et al. paper indicated cell loss in the hippocampus, including the CA3-CA1 synapses, but there was no cell loss in KO or cKO described in this paper.

4. Some speculation is warranted to discuss the possible mechanisms that lead from loss of NHE6 to activation of microglia, since it is what the authors conclude is happening.

Reviewer #2:

This is a strong and interesting manuscript which examines innovative new hypotheses that have broad relevance to Alzheimer's disease pathophysiology as well as potential new therapeutics. Pohlkamp, Herz and colleagues study the role NHE6 in several orthogonal studies related to production and deposition of amyloid plaques. The use of various different experimental approaches as well as the use of advance mouse genetics is a strength.

The authors demonstrate several important findings that are robustly supported by the data including: late loss of NHE6 leads to Purkinje cell degeneration; recycling defects in surface receptors relevant to AD and APOE4, namely APOER2 and Glu receptors is improved by deletion of NHE6; NHE6 KO restores Reelin enhancement of LTP inhibited by APOE4; and profound decrease in plaque deposition due to NHE6 mutation.

The data are well presented in general and compelling. There are many strengths. The PC findings are important in the field of Christianson Syndrome. The reductions in plaque load in the NHE6 null brains are VERY interesting. The mouse genetics, including the conditional mutation -- presentation of a new cKO NHE6 mouse, the humanized Abeta and APOE4 alleles, are truly elegant. Some of the experiments are uniquely supported by the prior findings from the lab relating to Reelin effects on endocytosis and trafficking and effects on LTP, and this is a very important strength. I do not see major weaknesses with the experiments as presented.

I believe that this work will have broad interest and this work and prior work of the Herz lab in the area of NHE6 as it may relate to therapeutics in AD is developing into a unique niche with potential strong impact in AD therapeutics.

Reviewer #3:

In this manuscript, Pohlkamp, Xian, Wang et al. investigated the role of the sodium-hydrogen exchanger NHE6 in synaptic plasticity and Aβ plaque load in a mouse model of Alzheimer's disease (AD) in the presence or absence of Apolipoprotein E4 (APOE4), a major genetic risk factor for sporadic AD. They initially report that NHE6 deletion causes cerebellar neurodegeneration. They find that genetic deletion of NHE6 alleviates impairments in reelin-induced synaptic plasticity in mice expressing human APOE4. The main novelty of this study is that NHE6 suppression significantly reduced amyloid plaque load in a mouse model of AD expressing humanized Aβ, either in the presence or absence of ApoE4. This is interesting, as it potentially opens new roads to understand and control amyloid pathology in the AD brain. Although the data are intriguing and relevant for the community, some issues need to be addressed so that conclusions are justified by data:

1) The leading hypothesis of this work is that APOE4 impairs synapse function through prolonged association with endosomes, thereby making brain cells vulnerable to AD-related pathological changes. However, the positive effects of NHE6 in a mouse model of Aβ accumulation occurs regardless of APOE4. This suggests that NHE6 may contribute to pathology by mechanisms other than APOE4-mediated retention of endosomal trafficking.

2) With the current data, it is not possible to exclude possible nonspecific effects resulting from NHE6 genetic deletion. Additional experiments to measure the endosomal pH would add support to the hypothesis.

3) The authors attribute reduced amyloid plaque load in NHE6-deficient APP KI mice to increased glial responses, which would promote plaque clearance. This is a very interesting hypothesis, but it is not supported by the experimental data reported in Supplemental Figure 6. Additional experimentation is needed to more thoroughly characterize astrocytic and microglial phenotypes caused by NHE6 genetic depletion in APP KI mice. Functional assays, including cytokine release, nitric oxide production (Griess reaction), and Aβ uptake experiments would be desired to strengthen these conclusions.

4) The authors demonstrate that global or conditional NHE6 deletion causes severe Purkinje cell loss in the mouse cerebellum (Figure 2). Although the authors included representative images of H&E staining indicating no gross histological abnormalities (Supplemental Figure 3), a more detailed investigation is required to assess neuronal survival in the hippocampus and cortex upon NHE6 suppression, given the relevance of these regions to AD pathology. Indeed, previous evidence (Xu et al., eNeuro, 2017) showed that NHE6 depletion leads to significant cortical and hippocampal atrophy, in addition to the cerebellum. Could the reductions in plaque load in NHE6 depleted mice (Figure 5, 6; Supplemental Figure 5) be somehow a reflection of neuronal loss? It is important that the authors discuss this issue in the manuscript.

5) Even though it is an important control assessment, data on cerebellar neurodegeneration (in addition to eventual new data on other brain regions to be included in the manuscript) could be moved to the supplementary file. Conversely, data on glial activation (in addition to eventual new data) could be moved to the main figures.

6) The authors should thoroughly revise their manuscript to make it more concise and straightforward. In particular, the Introduction and Discussion are excessively long and include several pieces of information that are not essential to understand the study.

7) It would be helpful to include a paragraph discussing the limitations of this study in the Discussion. In a revised Discussion section, it would also be relevant to comment on previous studies assessing potential behavioral, neuroinflammatory, and neurodegenerative phenotypes caused by NHE6 disruption (e.g. Xu et al., eNeuro, 2017; Petitjean et al., Pain, 2020) in mice. Authors should also comment on the apparently contradictory findings by Prasad and Rao, PNAS, 2018.

8) Statistical analyses need to be particularly revised for consistency. Data normality should be assessed, and suitable tests should be performed for each data set. Sample size description is often conflicting between graphs and figure legends, and the appropriate statistical tests have not been performed in all cases. For the sake of transparency, all bar graphs should be replaced by scatter dot plots.

9) A remaining question is whether NHE6 deletion does provide any cognitive benefit to APP KI mice.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "NHE6-Depletion Corrects ApoE4-Mediated Synaptic Impairments and Reduces Amyloid Plaque Load" for further consideration by eLife. Your revised article has been evaluated by Jonathan Cooper (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors.

The manuscript has been improved by the inclusion of new experiments and toning down of the text and conclusions drawn to better reflect what the data demonstrate. However, there are some remaining issues that need to be addressed, without additional experiments, as outlined below.

1. The new experiments that co-stained Abeta and Iba1 indicate that loss of NHE6 does not increase microglia around plaques, nor does it increase Abeta inside of microglia. The authors state that the results suggest increased efficiency of microglia in degrading Abeta. However, those results are also consistent with a lack of a role of microglia in the reduction of plaques in this case. Results from other studies have demonstrated that microglia can clear plaques, so this might be the most logical conclusion here as well, but no data to support this conclusion is presented in this manuscript. Therefore, the following is suggested:

1a. Further toning down of the manuscript; for example, at the end of the last Results section (page 20). The authors should remove the last statement that the mechanisms that lead to reduced plaque load in NHE6 deficient APP mice may involve increased catabolic rate, brought about by accelerated acidification and vesicular trafficking of early endosomes. There are two statements there for which the authors have not presented evidence – (1) that there is an increased catabolic rate, and (2) that accelerated acidification and endosomal trafficking increased catabolic rate. If the authors wish, they can discuss these possibilities in the discussion, and clearly state that these are speculations, but these statements should not be in the Results section.

1b. Clearer summary and discussion of the relative effects of NHE6 in neurons vs microglia or astrocytes.

1c. In the first paragraph of the discussion, the authors state that loss of NHE6 "suppressed" amyloid deposition. However, the data do not clearly distinguish whether there is reduction of deposition or enhancement of clearance. Perhaps saying that "loss of NHE6 reduces Abeta accumulation" may be more appropriate.

2. The Results sections describing the new data for (lack of) neuronal loss and co-staining of Abeta and Iba1/GFAP need to be edited and streamlined. There seems to be some inconsistency in the references to figures. Either the references are not in the right order, or the way they are referred to is not as streamlined as it could be.

3. The new discussion of the contradictory results related to Prasad and Rao 2018 highlights the difference between tissue culture versus genetically engineered mice as a possible reason underlying the discrepant results, but how about the difference of astrocyte vs neurons? Prasad an Rao overexpressed NHE6 in astrocytes, whereas the current manuscript manipulated primary neurons or mice. Such discussion would also enhance the readers' understanding of the relative roles of NHE6 in neurons vs microglia and astrocytes.

4. The authors state: "Xu et al. (2017) reported neuronal loss in the cortex and hippocampus of NHE6-KO mice, which we were able to reproduce in our germline NHE6-KO model (Supplemental Figure S3)."

Xu et al. (2017) examined tissue area. Xu et al. (2017) did not examine "neuronal loss" in cortex and hippocampus. The authors might agree that neuronal loss would reflect an observation wherein there is a measurable loss of neurons, ie counts of neurons are reduced. Instead, the studies reflect measurements of tissue area.

This underscores another new and important finding in the manuscript as stated: "We measured brain size, cortical thickness, hippocampal area, and CA1 thickness. In contrast to germline NHE6-KO mice (Supplemental Figure S3) none of the analyzed parameters differed significantly between NHE6- cKO and controls (Figure 5 E-I)."

These results could argue that the decrease tissue area in NHE6-KO, that is not seen in the NHE6-cKO, is due to undergrowth of cortex and hippocampus, ie the reduced tissue area in the NHE6-KO is a developmental effect due to loss of NHE6 during development. This would be predicted by the postnatal microcephaly seen in patients. This was also predicted by modeling in the Xu et al. paper: "The cerebrum data do fit an undergrowth-only model, with a similar degenerative rate as wild-type animals. The cerebellum strongly supports a mixed scenario of both undergrowth and enhanced neurodegeneration…" Further, Xu et al. describe that ventricle size is not vastly enlarged which might be predicted by neurodegeneration.

If the authors agree, the authors should consider editing their writing to reflect that Xu et al. did not look at "neuronal loss", and further, they may consider adding this interpretation of the difference between the tissue area in the NHE6-KO vs their NHE6-cKO to the discussion, ie that this may be a developmental effect.

5. Introduction: The authors may wish to consider shortening the first paragraph of the introduction, which is quite long (lines 2-43).

6. Abstract (line 13): change "amyloid" to "plaque load".

7. Experimental procedures:

7a. page 7, line 28: change "for primer" to "forward primer".

7b. page 8, lines 12-13: details (i.e. host, dilution, and catalog number) of antibodies raised against GFAP, IBA1, and Aβ should be provided.

7c. More details on the pH measurements should be provided. How many fields on each coverslip were quantified? How many vesicles per field were quantified? What was considered an experimental "n" (Supplemental Figure S1E-F)?

8. Results: In page 19 (lines 4-5) the conclusion is not related to the results presented in this paragraph. Results related to glial activation are only presented in the following paragraph.

9. Figure legends:

9a. Figure 2B: please specify the sample size.

9b. Figure 4: the phrase "Conditional knockout of NHE6 in ApoE4-KI mice attenuates reelin-enhanced long-term potentiation" is confusing and should be clarified.

9c. Supplemental Figure S1-E-F: statistical tests should be specified.

9d. Supplemental Figure S3: the reported sample sizes differ between the figure and the legend, which needs to be reconciled for accuracy.

10. Discussion: In page 20, lines 19-20: the phrase "hyper acidification of early endosomes occludes the effect of all ApoE forms on amyloid plaque formation" would be more accurate and easier to read if written as "hyper acidification of early endosomes prevents amyloid plaque formation independently of ApoE4".

https://doi.org/10.7554/eLife.72034.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

[…]

Weaknesses of this paper include:

1. The authors conclude that reducing NHE6 clears plaques by activating resident microglia, shifting them from a dormant state to a damage-associated activated state that phagocytoses Abeta plaques. However, there is no data presented to demonstrate this. In a supplemental figure, the authors show there are more Iba1-expressing microglia and GFAP-expressing astroctyes in APP mice and in APP/ApoE4KI mice in which NHE6 has been ablated, but this does not prove that this is the mechanism by which plaques are cleared.

We apologize for the overstatement. We agree, we have not evaluated whether NHE6 depletion causes a signature of damage-associated microglia. Thus, we have removed this comment from the manuscript (abstract).

2. The mechanisms underlying the increase in Iba1 and GFAP are not clear. The authors cite a previous paper from another group that demonstrated in their own NHE6 KO mice, there was an increase in GFAP and in activated microglia expressing CD68, which may relate to the cell loss in hippocampus and other brain regions documented in those mice. However, in the current study, the authors indicate that in their NHE6 KO lines, there is no overt cell loss. It is therefore unclear how reductions in NHE6 expression lead to microglial/astrocyte activation. This is an important point to work out, since the authors conclude that it is microglial activation that is responsible for the reduction in Abeta plaques.

We agree that identifying the mechanism how NHE6 depletion causes glial activation is crucial. We and others show that germline NHE6 depletion causes glial activation. Moreover, our current data suggest that genetic deletion of NHE6 in both germline and from adulthood on causes glial activation. Neuronal cell loss is a potential explanation for glial activation. We found cerebellar Purkinje cell loss in both of our NHE6 mutant lines. As stated in the old version of our manuscript we find “Normal Gross Anatomical Brain Structure in Both NHE6-KO and NHE6cKO Mice” (Supplementary Figure S3). To address whether neuronal loss occurs in the hippocampus or cortex, as described for NHE6-KO mice in Xu et al., 2017, we measured neuronal loss in both NHE6 mutant lines. Comparable to Xu et al., in the NHE6-KO line we detect a reduction in total brain area, HC area, cortical thickness and CA1 thickness. By contrast, in our NHE6-cKO;APP-KI;ApoE4-KI mice we do not observe any neuronal loss when compared to NHE6-floxed,APPNL-F,ApoE4-KI littermate controls; however, we detect similar glial activation in the NHE6-cKO;APPNL-F;ApoE4-KI mice as compared to the germline NHE6KO,APPNL-F mice. This suggests that the neuronal loss in the germline NHE6-KO model does not mediate glial activation. Lastly, we have removed the statement that the microglial activation is the reason why we detect Aβ reduction and included a discussion of our new findings.

3. What might be some of the underlying explanations be for the differences between the published NHE6-KO mice, which has fairly widespread cell loss, and the current KO mice generated in this paper, which did not exhibit noticeable cell loss in brain regions other than the cerebellum?

Our previous manuscript stated that there are no gross anatomical abnormalities in the NHE6KO mice. However, we appreciate the reviewer’s concerns as it prompted us to analyze neuronal loss in NHE6-KO versus NHE6-cKO mice. Besides Purkinje cell loss in both lines, and as stated above, we do detect cell loss in the hippocampus and cortex in our germline NHE6KO mouse model, but not in the tamoxifen induced NHE6-cKO mice.

4. There are a number of mechanistic links that have not been worked out, as indicated above. Until these links are identified and characterized, a number of the conclusions drawn by the authors are not yet supported.

We thank the reviewer for the constructive feedback. We have removed these conclusions.

Specific suggestions for authors:

1.To better assess whether the Iba1-expressing microglia are truly activated, CD68 should be stained. It would also be extremely compelling to stain for Abeta and demonstrate increased Abeta inside of microglia.

We thank the reviewer for this suggestion. Whereas we had too much non-specific background with the CD68 staining (see Author response image 1), we successfully co-stained Aβ with Iba1 and GFAP similar to others (Feng, W., et al., Alzheimers Res. Ther., 2020; Monasor, L., et al., ELife, 2020; Pomilio, C., et al., Geroscience, 2020; Parhizkar, S., Nat. Neurosci., et al., 2019; Zhon, L., et al., Nat. Commun., 2019). We did not detect a significant difference between genotypes in Aβ inside microglia. In addition, we quantified the amount of microglia and astrocytes surrounding plaques, with no significant differences between genotypes (NHE6-KO;APPNL-F and APPNL-F). As NHE6-KO mice show less plaque/Aβ-staining while having increased immunoreactivity for GFAP and Iba1, the fact that the proportion of microglia containing Aβ is equal to control indirectly suggests that the microglia are indeed more efficient in Aβ uptake and degradation. This hypothesis will be tested in future studies to evaluate Aβ uptake in primary microglia derived from NHE6-KO mice. We discussed our findings in the manuscript accordingly.

Author response image 1

2. In order to conclude that NHE6 ablation clears plaques BY activating microglia, the authors should deplete microglia and then see whether there is still an effect on plaque load. If there is, that would firmly support their hypothesis; but if eliminating microglia has no effect on plaques, it would suggest that there are other mechanisms at play.

We thank the reviewer for this suggestion. We are currently in the process of obtaining the animal approval for depleting the microglia in our mouse model. Future experiments will allow us to evaluate whether depleting the activated microglial affects the reduction of Aβ plaques.

3. Careful assessment of cell viability/death in the NHE6 mice (and related crosses) should be done. It is puzzling why the NHE6 KO lines here would show such differences in level of cell loss, relative to the study by Xu et al. It is an important point though, because (1) it could help provide a mechanism by which microglia get activated. (2) it is necessary to fully appreciate the LTP studies – the Xu et al. paper indicated cell loss in the hippocampus, including the CA3-CA1 synapses, but there was no cell loss in KO or cKO described in this paper.

As stated above, we evaluated cell loss in both the NHE6-KO and NHE6-cKO lines. In contrast to germline NHE6-KO mice, we did not detect cell loss in the cortex or hippocampus of the tamoxifen induced NHE6-cKO line, which was used in the electrophysiological studies. We agree with the reviewer that these novel data are crucial to better understand if NHE6-loss is a primary or secondary factor to induce glia activation.

4. Some speculation is warranted to discuss the possible mechanisms that lead from loss of NHE6 to activation of microglia, since it is what the authors conclude is happening.

We have added more potential mechanism in the discussion.

Reviewer #3:

In this manuscript, Pohlkamp, Xian, Wang et al. investigated the role of the sodium-hydrogen exchanger NHE6 in synaptic plasticity and Aβ plaque load in a mouse model of Alzheimer's disease (AD) in the presence or absence of Apolipoprotein E4 (APOE4), a major genetic risk factor for sporadic AD. They initially report that NHE6 deletion causes cerebellar neurodegeneration. They find that genetic deletion of NHE6 alleviates impairments in reelin-induced synaptic plasticity in mice expressing human APOE4. The main novelty of this study is that NHE6 suppression significantly reduced amyloid plaque load in a mouse model of AD expressing humanized Aβ, either in the presence or absence of ApoE4. This is interesting, as it potentially opens new roads to understand and control amyloid pathology in the AD brain. Although the data are intriguing and relevant for the community, some issues need to be addressed so that conclusions are justified by data:

1) The leading hypothesis of this work is that APOE4 impairs synapse function through prolonged association with endosomes, thereby making brain cells vulnerable to AD-related pathological changes. However, the positive effects of NHE6 in a mouse model of Aβ accumulation occurs regardless of APOE4. This suggests that NHE6 may contribute to pathology by mechanisms other than APOE4-mediated retention of endosomal trafficking.

We agree with the reviewer that NHE6 depletion plays a protective role in AD both by protecting against synaptic impairments in ApoE4-KI mice and Aβ toxicity in an Aβ overproducing mouse model. This may reflect a beneficial effect of endosomal compartment acidification through NHE6 depletion. Our current work and studies by others (Fagan, A.M., et al., Neurobio. of Dis. 2002) show that human Aβ-overproducing ApoE4-KI mice generate plaques at a much later age than mice with wildtype, mouse ApoE, but the mechanism is unknown. Since both of our mouse models, NHE6-KO and NHE6-cKO;ApoE4-KI show a comparable reduction in plaque load, this might be the result of a maximally accelerated early endosomal maturation and cargo transport in the absence of NHE6. We elaborated on this in topic in the discussion of our manuscript.

2) With the current data, it is not possible to exclude possible nonspecific effects resulting from NHE6 genetic deletion. Additional experiments to measure the endosomal pH would add support to the hypothesis.

We agree with the reviewer’s concern and addressed this in the discussion accordingly.

3) The authors attribute reduced amyloid plaque load in NHE6-deficient APP KI mice to increased glial responses, which would promote plaque clearance. This is a very interesting hypothesis, but it is not supported by the experimental data reported in Supplemental Figure 6. Additional experimentation is needed to more thoroughly characterize astrocytic and microglial phenotypes caused by NHE6 genetic depletion in APP KI mice. Functional assays, including cytokine release, nitric oxide production (Griess reaction), and Aβ uptake experiments would be desired to strengthen these conclusions.

We thank the reviewer for this valuable feedback. In our revised manuscript, we evaluated whether there is a change of microglial Ab content in the NHE6 depletion mouse model. We also quantified the immunoreactivity of Iba1 and GFAP in plaque areas. We found no change between NHE6-KO or control littermate APPNL-F controls when we co-stained with Aβ and Iba1 (microglia) or GFAP (astrocyte). However, when considering a massively reduced Aβ signal in NHE6-KO brains overall, yet the proportion of microglia containing Aβ is comparable to control, this indirectly indicates that NHE6 deficient microglia are more efficient in Aβ uptake and degradation. We agree with the reviewers that future studies will be required to evaluate Aβ uptake in primary microglia derived from NHE6-KO mice to properly conclude that the reduction of Aβ is mediated by enhance glial activation. Thus, we have adjusted our conclusions in the manuscript accordingly.

4) The authors demonstrate that global or conditional NHE6 deletion causes severe Purkinje cell loss in the mouse cerebellum (Figure 2). Although the authors included representative images of H&E staining indicating no gross histological abnormalities (Supplemental Figure 3), a more detailed investigation is required to assess neuronal survival in the hippocampus and cortex upon NHE6 suppression, given the relevance of these regions to AD pathology. Indeed, previous evidence (Xu et al., eNeuro, 2017) showed that NHE6 depletion leads to significant cortical and hippocampal atrophy, in addition to the cerebellum. Could the reductions in plaque load in NHE6 depleted mice (Figure 5, 6; Supplemental Figure 5) be somehow a reflection of neuronal loss? It is important that the authors discuss this issue in the manuscript.

We thank the reviewer for this suggestion. We have now measured brain area, hippocampal area, cortical and CA1 thickness. Comparable to Xu et al., we detect a reduction in total brain area, HC area, cortical thickness and CA1 thickness. Contrary, in our NHE6-cKO;APPNFL;ApoE4-KI mice we do not see any neuronal loss; however, we detect similar plaque reduction and glial activation in the NHE6-KO;APPNL-F mice. These findings suggest that the neuronal loss does not mediate the reduction in plaque load or glial activation. We discussed our findings in our manuscript accordingly.

5) Even though it is an important control assessment, data on cerebellar neurodegeneration (in addition to eventual new data on other brain regions to be included in the manuscript) could be moved to the supplementary file. Conversely, data on glial activation (in addition to eventual new data) could be moved to the main figures.

We thank the reviewer for the suggestion. We included new data on neuronal loss in additional brain regions of the NHE6 mutant mice. As specific findings on germline NHE6-KO mice are already published by others (and now reproduced by us), we decided to keep our results on NHE6-KO in the supplements, while keeping the novel results on NHE6-cKO in the main figures. Our novel data on glia and Aβ co-labeling are now included in the main manuscript, Figure 7.

6) The authors should thoroughly revise their manuscript to make it more concise and straightforward. In particular, the Introduction and Discussion are excessively long and include several pieces of information that are not essential to understand the study.

We thank the reviewer for this comment and shortened the introduction. While adding several points to our discussion, based on reviewers’ suggestions and novel findings, we also shortened it by removing the paragraph addressing NHE6 C-terminal interactions, since it is not relevant to understand the study.

7) It would be helpful to include a paragraph discussing the limitations of this study in the Discussion. In a revised Discussion section, it would also be relevant to comment on previous studies assessing potential behavioral, neuroinflammatory, and neurodegenerative phenotypes caused by NHE6 disruption (e.g. Xu et al., eNeuro, 2017; Petitjean et al., Pain, 2020) in mice. Authors should also comment on the apparently contradictory findings by Prasad and Rao, PNAS, 2018.

We thank the reviewer for this suggestion and have added new paragraphs to our discussion that also address limitations of our study. We also discussed the findings by Prasad and Rao (Prasad, H. and Rao, R., PNAS 2018).

8) Statistical analyses need to be particularly revised for consistency. Data normality should be assessed, and suitable tests should be performed for each data set. Sample size description is often conflicting between graphs and figure legends, and the appropriate statistical tests have not been performed in all cases. For the sake of transparency, all bar graphs should be replaced by scatter dot plots.

We thank the reviewer for pointing this out. We carefully went through the statistics and the numbers. Corrections have been made for figure legends 6, S5, and S6. Scatter dot plots have been provided for Figure 3 and 7.

9) A remaining question is whether NHE6 deletion does provide any cognitive benefit to APP KI mice.

We agree with the reviewer that behavioral insights would be compelling. However, APPNL-F mice do not show behavioral abnormalities until at least 18 months of age (Saito et al., 2014). In addition, when we bred APPNL-F with ApoE4 and ApoE3 mice, we were not able to detect differences in the Morris Water Maze test between ApoE genotype, either. Thus, we are unable to determine whether NHE6 depletion provides any cognitive benefits in the APPNL-F mouse line. We addressed this in our revised discussion.

[Editors’ note: what follows is the authors’ response to the second round of review.]

The manuscript has been improved by the inclusion of new experiments and toning down of the text and conclusions drawn to better reflect what the data demonstrate. However, there are some remaining issues that need to be addressed, without additional experiments, as outlined below.

1. The new experiments that co-stained Abeta and Iba1 indicate that loss of NHE6 does not increase microglia around plaques, nor does it increase Abeta inside of microglia. The authors state that the results suggest increased efficiency of microglia in degrading Abeta. However, those results are also consistent with a lack of a role of microglia in the reduction of plaques in this case. Results from other studies have demonstrated that microglia can clear plaques, so this might be the most logical conclusion here as well, but no data to support this conclusion is presented in this manuscript. Therefore, the following is suggested:

1a. Further toning down of the manuscript; for example, at the end of the last Results section (page 20). The authors should remove the last statement that the mechanisms that lead to reduced plaque load in NHE6 deficient APP mice may involve increased catabolic rate, brought about by accelerated acidification and vesicular trafficking of early endosomes. There are two statements there for which the authors have not presented evidence – (1) that there is an increased catabolic rate, and (2) that accelerated acidification and endosomal trafficking increased catabolic rate. If the authors wish, they can discuss these possibilities in the discussion, and clearly state that these are speculations, but these statements should not be in the Results section.

We agree that this last statement should be removed from our Results section and added to the discussion (Page 24).

1b. Clearer summary and discussion of the relative effects of NHE6 in neurons vs microglia or astrocytes.

We have added a detailed discussion (Page 25) on the effects of NHE6 in various cell types (neurons, microglia or astrocytes).

1c. In the first paragraph of the discussion, the authors state that loss of NHE6 "suppressed" amyloid deposition. However, the data do not clearly distinguish whether there is reduction of deposition or enhancement of clearance. Perhaps saying that "loss of NHE6 reduces Abeta accumulation" may be more appropriate.

We have adjusted this statement in our discussion (Page 20).

2. The Results sections describing the new data for (lack of) neuronal loss and co-staining of Abeta and Iba1/GFAP need to be edited and streamlined. There seems to be some inconsistency in the references to figures. Either the references are not in the right order, or the way they are referred to is not as streamlined as it could be.

We apologize for any confusion and have made the requested modifications to help streamline the figures and text, which is located on page 18 and 19.

3. The new discussion of the contradictory results related to Prasad and Rao 2018 highlights the difference between tissue culture versus genetically engineered mice as a possible reason underlying the discrepant results, but how about the difference of astrocyte vs neurons? Prasad an Rao overexpressed NHE6 in astrocytes, whereas the current manuscript manipulated primary neurons or mice. Such discussion would also enhance the readers' understanding of the relative roles of NHE6 in neurons vs microglia and astrocytes.

We apologize for any confusion, and we made corrections to this paragraph. Previously, we referred to astrocytes when in fact Prasad and Rao used HEK293 cells (Prasad and Rao, 2015) to look at Ab generation. We have added additional text to explain the discrepant results (page 23) and included section for the readers about the relative roles of NHE6 in neurons versus microglia and astrocytes (Page 25, see Reviewer Comment 1b).

4. The authors state: "Xu et al. (2017) reported neuronal loss in the cortex and hippocampus of NHE6-KO mice, which we were able to reproduce in our germline NHE6-KO model (Supplemental Figure S3)."

Xu et al. (2017) examined tissue area. Xu et al. (2017) did not examine "neuronal loss" in cortex and hippocampus. The authors might agree that neuronal loss would reflect an observation wherein there is a measurable loss of neurons, ie counts of neurons are reduced. Instead, the studies reflect measurements of tissue area.

This underscores another new and important finding in the manuscript as stated: "We measured brain size, cortical thickness, hippocampal area, and CA1 thickness. In contrast to germline NHE6-KO mice (Supplemental Figure S3) none of the analyzed parameters differed significantly between NHE6- cKO and controls (Figure 5 E-I)."

These results could argue that the decrease tissue area in NHE6-KO, that is not seen in the NHE6-cKO, is due to undergrowth of cortex and hippocampus, ie the reduced tissue area in the NHE6-KO is a developmental effect due to loss of NHE6 during development. This would be predicted by the postnatal microcephaly seen in patients. This was also predicted by modeling in the Xu et al. paper: "The cerebrum data do fit an undergrowth-only model, with a similar degenerative rate as wild-type animals. The cerebellum strongly supports a mixed scenario of both undergrowth and enhanced neurodegeneration…" Further, Xu et al. describe that ventricle size is not vastly enlarged which might be predicted by neurodegeneration.

If the authors agree, the authors should consider editing their writing to reflect that Xu et al. did not look at "neuronal loss", and further, they may consider adding this interpretation of the difference between the tissue area in the NHE6-KO vs their NHE6-cKO to the discussion, ie that this may be a developmental effect.

We thank the reviewer for this valuable discussion of our findings and the important point clarifying Xu et al. We have revised discussion accordingly and included detailed discussion of the Xu et al. findings in the context of our results (Page 18, 19, and 25).

5. Introduction: The authors may wish to consider shortening the first paragraph of the introduction, which is quite long (lines 2-43).

We appreciate this comment and we have accordingly introduced line breaks into the first paragraph (Page 3).

6. Abstract (line 13): change "amyloid" to "plaque load".

We’ve changed this text in the abstract.

7. Experimental procedures:

7a. page 7, line 28: change "for primer" to "forward primer".

7b. page 8, lines 12-13: details (i.e. host, dilution, and catalog number) of antibodies raised against GFAP, IBA1, and Aβ should be provided.

7c. More details on the pH measurements should be provided. How many fields on each coverslip were quantified? How many vesicles per field were quantified? What was considered an experimental "n" (Supplemental Figure S1E-F)?

We have added these experimental details to the experimental procedures.

8. Results: In page 19 (lines 4-5) the conclusion is not related to the results presented in this paragraph. Results related to glial activation are only presented in the following paragraph.

We have adjusted the text accordingly.

9. Figure legends:

9a. Figure 2B: please specify the sample size.

9b. Figure 4: the phrase "Conditional knockout of NHE6 in ApoE4-KI mice attenuates reelin-enhanced long-term potentiation" is confusing and should be clarified.

9c. Supplemental Figure S1-E-F: statistical tests should be specified.

9d. Supplemental Figure S3: the reported sample sizes differ between the figure and the legend, which needs to be reconciled for accuracy.

We apologize for the oversight and typos. We have adjusted the text accordingly.

10. Discussion: In page 20, lines 19-20: the phrase "hyper acidification of early endosomes occludes the effect of all ApoE forms on amyloid plaque formation" would be more accurate and easier to read if written as "hyper acidification of early endosomes prevents amyloid plaque formation independently of ApoE4".

We have changed this text.

https://doi.org/10.7554/eLife.72034.sa2

Article and author information

Author details

  1. Theresa Pohlkamp

    1. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    2. Center for Translational Neurodegeneration Research, Dallas, United States
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing
    Contributed equally with
    Xunde Xian and Connie H Wong
    For correspondence
    ThePohlkamp@gmail.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3923-1917
  2. Xunde Xian

    1. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    2. Center for Translational Neurodegeneration Research, Dallas, United States
    3. Institute of Cardiovascular Sciences and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Peking University, Beijing, China
    Contribution
    Conceptualization, Investigation, Methodology, Resources, Validation, Visualization
    Contributed equally with
    Theresa Pohlkamp and Connie H Wong
    Competing interests
    Inventor of Patent: https://patents.google.com/patent/US20110136832A1/en
  3. Connie H Wong

    1. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    2. Center for Translational Neurodegeneration Research, Dallas, United States
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review and editing
    Contributed equally with
    Theresa Pohlkamp and Xunde Xian
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6452-7966
  4. Murat S Durakoglugil

    1. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    2. Center for Translational Neurodegeneration Research, Dallas, United States
    Contribution
    Formal analysis, Investigation, Methodology, Resources, Validation, Visualization
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4483-8166
  5. Gordon Chandler Werthmann

    1. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    2. Center for Translational Neurodegeneration Research, Dallas, United States
    Contribution
    Formal analysis, Investigation, Methodology, Validation
    Competing interests
    No competing interests declared
  6. Takaomi C Saido

    Laboratory for Proteolytic Neuroscience, Riken Center for Brain Science, Wako, Japan
    Contribution
    Resources
    Competing interests
    No competing interests declared
  7. Bret M Evers

    Center for Translational Neurodegeneration Research, Dallas, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5686-0315
  8. Charles L White III

    Pathology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Resources
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3870-2804
  9. Jade Connor

    1. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    2. Center for Translational Neurodegeneration Research, Dallas, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  10. Robert E Hammer

    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  11. Joachim Herz

    1. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    2. Center for Translational Neurodegeneration Research, Dallas, United States
    3. Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, United States
    4. Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review and editing
    For correspondence
    joachim.herz@utsouthwestern.edu
    Competing interests
    Inventor of Patent: https://patents.google.com/patent/US20110136832A1/en
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8506-3400

Funding

National Institutes of Health (HL063762)

  • Joachim Herz

BrightFocus Foundation (A20135245)

  • Joachim Herz

National Institute on Aging (1F31AG067708-01)

  • Connie H Wong

Darrell K Royal Research Fund

  • Murat S Durakoglugil

Harrington Discovery Institute

  • Joachim Herz

Circle of Friends Pilot Synergy

  • Joachim Herz

Blue Field Project to Cure FTD

  • Joachim Herz

National Institutes of Health (NS093382)

  • Joachim Herz

National Institutes of Health (NS108115)

  • Joachim Herz

National Institutes of Health (AG053391)

  • Joachim Herz

BrightFocus Foundation (A2016396S)

  • Joachim Herz

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by NIH grants R37 HL063762, R01 NS093382, R01 NS108115, and RF1 AG053391 to JH and 1F31 AG067708-01 to CHW as well as funding from the Darrell K Royal Research Fund to MD. While this work was ongoing, JH was further supported by the Bright Focus Foundation (A20135245) and (A2016396S); Harrington Discovery Institute; and Circle of Friends Pilot Synergy Grant; and the Blue Field Project to Cure FTD. We are indebted Rebekah Hewitt, Barsha Subbha, Huichuan Reyna, Issac Rocha, Tamara Terrones, Emily Boyle, Alisa Gilloon, Travis Wolff, and Eric Hall for their excellent technical assistance. We thank Dr Yuan Yang for creating the NHE6-FLAG plasmid and the UTSW Whole Brain Microscopy Facility (WBMF) in the Department of Neurology and Neurotherapeutics for assistance with slide scanning. The WBMF is supported by the Texas Institute for Brain Injury and Repair (TIBIR). John Shelton and the UT Southwestern’s Histopathology Core provided help with paraffin sectioning as well as H&E and Thioflavin S staining. We thank Wolfgang Scholz for providing EMD87580.

Ethics

All animal procedures were performed according to the approved guidelines (Animal Welfare Assurance Number D16-00296) for Institutional Animal Care and Use Committee (IACUC) at the University of Texas Southwestern Medical Center at Dallas.

Senior Editor

  1. Jonathan A Cooper, Fred Hutchinson Cancer Research Center, United States

Reviewing Editor

  1. Jeannie Chin, Baylor College of Medicine, United States

Reviewer

  1. Eric Morrow, Brown University

Publication history

  1. Preprint posted: March 23, 2021 (view preprint)
  2. Received: July 7, 2021
  3. Accepted: September 19, 2021
  4. Accepted Manuscript published: October 7, 2021 (version 1)
  5. Version of Record published: October 26, 2021 (version 2)

Copyright

© 2021, Pohlkamp et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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