Author response: NHE6 depletion corrects ApoE4-mediated synaptic impairments and reduces amyloid plaque load (2021)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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). Figure 1 Download asset Open asset 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). Figure 2 Download asset Open asset 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). Figure 3 Download asset Open asset 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). Figure 4 Download asset Open asset 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 recycling could be by endosomal acidification induced by knockdown of Slc9a6 or by the (Xian et al., 2018). To further a effect caused by the inhibition of or by we applied this assay on neurons isolated from Slc9a6- (Figure Apoer2 recycling was in Slc9a6- neurons treated with ApoE4 (Figure We previously reported that the of ApoE3 to neurons also Apoer2 trafficking to a was also in Slc9a6- neurons (Figure In addition, genetic loss of NHE6 the ApoE4-impaired surface expression of and NMDA receptor (Figure disruption of NHE6 synaptic Reelin resistance in ApoeAPOE4 mice Reelin can in hippocampal field of not ApoeAPOE4 brain (Chen et al., 2010). We previously showed that this Reelin resistance in ApoeAPOE4 was by inhibition (Xian et al., 2018). To investigate loss of NHE6 also restores synaptic plasticity in the presence of ApoE4, we performed hippocampal field on Slc9a6fl;CAG-CreERT2 mice bred to or ApoeAPOE4 mice. To avoid effects of NHE6 deficiency embryonic development (Ouyang et al., NHE6/Slc9a6 gene disruption was induced at by tamoxifen et al., was performed after NHE6 depletion in mice (Slc9a6fl;CAG-CreERT2, Figure and mice expressing human ApoE3 or ApoE4 as and Figure and For field hippocampal were with Reelin as described et al., 2005; et al., 2010; Durakoglugil et al., 2009; et al., 2002). genetic loss of NHE6 in a reduction of the ability of Reelin to in mice Figure and to and By contrast, as reported previously (Chen et al., hippocampal from ApoeAPOE4 mice were to by Reelin (Figure and resistance was when NHE6 was in ApoeAPOE4 Reelin application (Figure and in to a as in the mice (Figure was and were generated by the measured at increasing against the were found (Figure and Figure Download asset Open asset of conditional sodium-hydrogen exchanger 6 (NHE6) knockout on synaptic knockout of NHE6 restores in ApoeAPOE4 mice. Reelin of in not ApoeAPOE4 (C, control mice. Slc9a6 deficiency in mice caused a reduction in that it is not significantly different from the control Importantly, in mice Reelin was to were prepared from double mutant mice with human or ApoeAPOE4 with Slc9a6 conditional knockout mice (Slc9a6fl;CAG-CreERT2, tamoxifen at field were performed in treated with or without was performed after min of are shown in each before and min after Quantitative analysis of at time as of and ApoeAPOE4 mice with or without Slc9a6fl mice includes both female Slc9a6fl/fl and male mice. mice are for or All data are expressed as mean ± SEM. for each group and treatment are indicated in panels *p < 0.05. Statistical analysis was performed using Student’s t-test. inhibition or NHE6 knockdown not alter β-CTF in vitro of APP by γ-secretase and the BACE1 the short neurotoxic by BACE1 results in a fragment which is further by γ-secretase to the soluble Aβ peptide. BACE1 of APP in the complex, on the cell and after endocytosis in endosomes et al., 1994; et al., 1999). It has been reported that BACE1 activity with lower pH et al., 2002). In a in vitro in an cell line APP and NHE6 led to a reduction of Aβ and knockdown of NHE6/Slc9a6 in an increase in Aβ and To investigate NHE6/Slc9a6 deficiency to APP by BACE1 in neurons, we used primary neurons from an Alzheimer’s disease (AD) mouse model that human APP with the et al., were treated with the or with against NHE6/Slc9a6 in the presence or absence of the γ-secretase β-CTF was using the against Aβ residues (Figure of γ-secretase in neurons β-CTF accumulation, treatment with did not alter the amount of β-CTF in the cell lysates (Figure NHE6 knockdown using also had effect on the amount of β-CTF (Figure We that NHE6 inhibition is to increase Aβ under Figure 6 Download asset Open asset exchanger inhibition or sodium-hydrogen exchanger 6 (NHE6) knockdown not alter β-site amyloid precursor protein cleaving enzyme 1 (BACE1) activity in primary neurons. inhibition by or knockdown of Slc9a6 (NHE6) did not alter BACE1 activity in primary neurons of mice (A) primary neurons were treated with γ-secretase and/or ApoE4 and harvested for immunoblotting against C-terminal fragment of APP β-Actin was as loading control. shows the of = 3 experiments. (B) Primary neurons of mice were infected with lentivirus for expression against or a control at At neurons were treated with and harvested for immunoblotting against NHE6 and β-CTF on was as loading control. shows the of = 6 experiments. All data are expressed as mean ± SEM. Statistical analysis was performed using Student’s t-test. = not NHE6 deficiency reduces Aβ plaque load in human knockin mice To further the effect of NHE6 deficiency on Aβ pathology in vivo, we bred AppNL-F mice (Saito et al., 2014) to our germline NHE6 KO line In these AppNL-F the Aβ has been and the early onset AD and = and the = were also resulting in Aβ regulation of APP expression. to the effect of Aβ while APP expression under the control of the and control AppNL-F littermates were to 1 brains were harvested and analyzed by and to plaque and Aβ did not between brain hippocampal and CA1 were as described previously et al., 2017; Figure were more frequent in Slc9a6 wildtype than Slc9a6- mice. To further investigate and quantify plaque we analyzed the same brains after (Figure We found an 80% reduction of in Slc9a6- mice when compared to littermate (Figure against Aβ showed the same reduction (Figure In addition, we analyzed soluble and and Aβ in brain lysates of mice and their control The amount of Aβ and was reduced in mice by approximately when compared to their control littermates (Figure The reduction in soluble Aβ was not in Slc9a6- lysates Figure with 3 Download asset Open asset Sodium-hydrogen exchanger 6 (NHE6) deficiency plaque in both AppNL-F and mice. AppNL-F and control AppNL-F mice were analyzed for plaque deposition at an age of was performed to plaques. were found more in the control AppNL-F mice (left panel in of the are shown in the two The plaque load between Slc9a6- mice and control littermates was compared and (B) In the Slc9a6- the plaque number was when compared to