Loss of Christianson Syndrome Na+/H+ Exchanger 6 (NHE6) Causes Abnormal Endosome Maturation and Trafficking Underlying Lysosome Dysfunction in Neurons.

Loss-of-function mutations in endosomal Na+/H+ exchanger 6 (NHE6) cause the X-linked neurologic disorder Christianson syndrome. Patients exhibit symptoms associated with both neurodevelopmental and neurodegenerative abnormalities. While loss of NHE6 has been shown to overacidify the endosome lumen, and is associated with endolysosome neuropathology, NHE6-mediated mechanisms in endosome trafficking and lysosome function have been understudied. Here, we show that NHE6-null mouse neurons demonstrate worsening lysosome function with time in culture, likely as a result of defective endosome trafficking. NHE6-null neurons exhibit overall reduced lysosomal proteolysis despite overacidification of the endosome and lysosome lumen. Akin to Nhx1 mutants in Saccharomyces cerevisiae, we observe decreased endosome-lysosome fusion in NHE6-null neurons. Also, we find premature activation of pH-dependent cathepsin D (CatD) in endosomes. While active CatD is increased in endosomes, CatD activation and CatD protein levels are reduced in the lysosome. Protein levels of another mannose 6-phosphate receptor (M6PR)-dependent enzyme, ß-N-acetylglucosaminidase, were also decreased in lysosomes of NHE6-null neurons. M6PRs accumulate in late endosomes, suggesting defective M6PR recycling and retromer function in NHE6-null neurons. Finally, coincident with decreased endosome-lysosome fusion, using total internal reflection fluorescence, we also find a prominent increase in fusion between endosomal multivesicular bodies and the plasma membrane, indicating enhanced exosome secretion from NHE6-null neurons. In summary, in addition to overacidification of endosomes and lysosomes, loss of NHE6 leads to defects in endosome maturation and trafficking, including enhanced exosome release, contributing to lysosome deficiency and potentially leading to neurodegenerative disease.SIGNIFICANCE STATEMENT Loss-of-function mutations in the endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome, an X-linked neurologic disorder. Loss of NHE6 has been shown to overacidify endosomes; however, endosome trafficking mechanisms have been understudied, and the mechanisms leading to neurodegeneration are largely unknown. In NHE6-null mouse neurons in vitro, we find worsening lysosome function with days in culture. Notably, pH-dependent lysosome enzymes, such as cathepsin D, have reduced activity in lysosomes yet increased, precocious activity in endosomes in NHE6-null neurons. Further, endosomes show reduced fusion to lysosomes, and increased fusion to the plasma membrane with increased exosome release. This study identifies new mechanisms involving defective endosome maturation and trafficking that impair lysosome function in Christianson syndrome, likely contributing to neurodegeneration.

An indisputable role of NHE8 in mucosal protection.

The loss of the intestinal Na+/H+ exchanger isoform 8 (NHE8) results in an ulcerative colitis-like condition with reduction of mucin production and dysbiosis, indicating that NHE8 plays an important role in intestinal mucosal protection. The aim of this study was to investigate the potential rebalance of the altered microbiota community of NHE8-deficient mice via fecal microbiota transplantation (FMT) and feeding probiotic VSL#3. We also aimed to stimulate mucin production by sodium butyrate administration via enema. Data from 16S rRNA sequencing showed that loss of NHE8 contributes to colonic microbial dysbiosis with reduction of butyrate-producing bacteria. FMT increased bacterial adhesion in the colon in NHE8 knockout (NHE8KO) mice. Periodic-acid Schiff reagent (PAS) stain and quantitative PCR showed no changes in mucin production during FMT. In mice treated with the probiotic VSL#3, a reduction of Lactobacillus and segmented filamentous bacteria (SFB) in NHE8KO mouse colon was detected and an increase in goblet cell theca was observed. In NHE8KO mice receiving sodium butyrate (NaB), 1 mM NaB stimulated Muc2 expression without changing goblet cell theca, but 10 mM NaB induced a significant reduction of goblet cell theca without altering Muc2 expression. Furthermore, 5 mM and 10 mM NaB-treated HT29-MTX cells displayed increased apoptosis, while 0.5 mM NaB stimulated Muc2 gene expression. These data showed that loss of NHE8 leads to dysbiosis with reduction of butyrate-producing bacteria and FMT and VSL#3 failed to rebalance the microbiota in NHE8KO mice. Therefore, FMT, VSL#3, and NaB are not able to restore mucin production in the absence of NHE8 in the intestine.NEW & NOTEWORTHY Loss of Na+/H+ exchanger isoform 8 (NHE8), a Slc9 family of exchanger that contributes to sodium uptake, cell volume regulation, and intracellular pH homeostasis, resulted in dysbiosis with reduction of butyrate-producing bacteria and decrease of Muc2 production in the intestine in mice. Introducing fecal microbiota transplantation (FMT) and VSL#3 in NHE8 knockout (NHE8KO) mice failed to rebalance the microbiota in these mice. Furthermore, administration of FMT, VSL#3, and sodium butyrate was unable to restore mucin production in the absence of NHE8 in the intestine.

Deficient Active Transport Activity in Healing Mucosa After Mild Gastric Epithelial Damage.

BACKGROUND: Peptic ulcers recur, suggesting that ulcer healing may leave tissue predisposed to subsequent damage. In mice, we have identified that the regenerated epithelium found after ulcer healing will remain abnormal for months after healing. AIM: To determine whether healed gastric mucosa has altered epithelial function, as measured by electrophysiologic parameters. METHOD: Ulcers were induced in mouse gastric corpus by serosal local application of acetic acid. Thirty days or 8 months after ulcer induction, tissue was mounted in an Ussing chamber. Transepithelial electrophysiologic parameters (short-circuit current, Isc. resistance, R) were compared between the regenerated healed ulcer region and the non-ulcerated contralateral region, in response to luminal hyperosmolar NaCl challenge (0.5 M). RESULTS: In unperturbed stomach, luminal application of hyperosmolar NaCl transiently dropped Isc followed by gradual recovery over 2 h. Compared to the starting baseline Isc, percent Isc recovery was reduced in 30-day healing mucosa, but not at 8 months. Prior to NaCl challenge, a lower baseline Isc was observed in trefoil factor 2 (TFF2) knockout (KO) versus wild type (WT), with no Isc recovery in either non-ulcerated or healing mucosa of KO. Inhibiting Na/H exchanger (NHE) transport in WT mucosa inhibited Isc recovery in response to luminal challenge. NHE2-KO baseline Isc was reduced versus NHE2-WT. In murine gastric organoids, NHE inhibition slowed recovery of intracellular pH and delayed the repair of photic induced damage. CONCLUSION: Healing gastric mucosa has deficient electrophysiological recovery in response to hypertonic NaCl. TFF2 and NHE2 contribute to Isc regulation, and the recovery and healing of transepithelial function.

NHE8 Deficiency Promotes Colitis-Associated Cancer in Mice via Expansion of Lgr5-Expressing Cells.

Background & Aims: Lgr5 overexpression has been detected in colorectal cancers (CRCs), including some cases of colitis-associated CRCs. In colitis-associated CRCs, chronic inflammation is a contributing factor in carcinogenesis. We recently reported that intestinal Na+/H+ exchanger isoform 8 (NHE8) plays an important role in intestinal mucosal protection and that loss of NHE8 expression results in an ulcerative colitis-like condition. Therefore, we hypothesized that NHE8 may be involved in the development of intestinal tumors. Methods: We assessed NHE8 expression in human CRCs by immunohistochemistry and studied tumor burden in NHE8 knockout (KO) mice using an azoxymethane/dextran sodium sulfate colon cancer model. We also evaluated cell proliferation in HT29NHE8KO cells and assessed tumor growth in NOD scid gamma (NSG) mice xenografted with HT29NHE8KO cells. To verify if a relationship exists between Lgr5 and NHE8 expression, we analyzed Lgr5 expression in NHE8KO mice by polymerase chain reaction and in situ hybridization. Lgr5 expression and cell proliferation in the absence of NHE8 were confirmed in colonic organoid cultures. The expression of ß-catenin and c-Myc also were analyzed to evaluate Wnt/ß-catenin activation. Results: NHE8 was undetectable in human CRC tissues. Although only 9% of NHE8 wild-type mice showed tumorigenesis in the azoxymethane/dextran sodium sulfate colon cancer model, almost 10 times more NHE8KO mice (89%) developed tumors. In the absence of NHE8, a higher colony formation unit was discovered in HT29NHE8KO cells. In NSG mice, larger tumors developed at the site where HT29NHE8KO cells were injected compared with HT29NHE8 wild type cells. Furthermore, NHE8 deficiency resulted in increased Lgr5 expression in the colon, in HT29-derived tumors, and in colonoids. The absence of NHE8 also increased Wnt/ß-catenin activation. Conclusions: NHE8 might be an intrinsic factor that regulates Wnt/ß-catenin in the intestine.

A mouse model of autism implicates endosome pH in the regulation of presynaptic calcium entry.

Psychoactive compounds such as chloroquine and amphetamine act by dissipating the pH gradient across intracellular membranes, but the physiological mechanisms that normally regulate organelle pH remain poorly understood. Interestingly, recent human genetic studies have implicated the endosomal Na+/H+ exchanger NHE9 in both autism spectrum disorders (ASD) and attention deficit hyperactivity disorder (ADHD). Plasma membrane NHEs regulate cytosolic pH, but the role of intracellular isoforms has remained unclear. We now find that inactivation of NHE9 in mice reproduces behavioral features of ASD including impaired social interaction, repetitive behaviors, and altered sensory processing. Physiological characterization reveals hyperacidic endosomes, a cell-autonomous defect in glutamate receptor expression and impaired neurotransmitter release due to a defect in presynaptic Ca2+ entry. Acute inhibition of synaptic vesicle acidification rescues release but without affecting the primary defect due to loss of NHE9.

Both NHERF3 and NHERF2 are necessary for multiple aspects of acute regulation of NHE3 by elevated Ca2+, cGMP, and lysophosphatidic acid.

The intestinal epithelial brush border Na+/H+ exchanger NHE3 accounts for a large component of intestinal Na absorption. NHE3 is regulated during digestion by signaling complexes on its COOH terminus that include the four multi-PDZ domain-containing NHERF family proteins. All bind to NHE3 and take part in different aspects of NHE3 regulation. Because the roles of each NHERF appear to vary on the basis of the cell model or intestinal segment studied and because of our recent finding that a NHERF3-NHERF2 heterodimer appears important for NHE3 regulation in Caco-2 cells, we examined the role of NHERF3 and NHERF2 in C57BL/6 mouse jejunum using homozygous NHERF2 and NHERF3 knockout mice. NHE3 activity was determined with two-photon microscopy and the dual-emission pH-sensitive dye SNARF-4F. The jejunal apical membrane of NHERF3-null mice appeared similar to wild-type (WT) mice in surface area, microvillus number, and height, which is similar to results previously reported for jejunum of NHERF2-null mice. NHE3 basal activity was not different from WT in either NHERF2- or NHERF3-null jejunum, while d-glucose-stimulated NHE3 activity was reduced in NHERF2, but similar to WT in NHERF3 KO. LPA stimulation and UTP (elevated Ca2+) and cGMP inhibition of NHE3 were markedly reduced in both NHERF2- and NHERF3-null jejunum. Forskolin inhibited NHE3 in NHERF3-null jejunum, but the extent of inhibition was reduced compared with WT. The forskolin inhibition of NHE3 in NHERF2-null mice was too inconsistent to determine whether there was an effect and whether it was altered compared with the WT response. These results demonstrate similar requirement for NHERF2 and NHERF3 in mouse jejunal NHE3 regulation by LPA, Ca2+, and cGMP. The explanation for the similarity is not known but is consistent with involvement of a brush-border NHERF3-NHERF2 heterodimer or sequential NHERF-dependent effects in these aspects of NHE3 regulation. NEW & NOTEWORTHY NHERF2 and NHERF3 are apical membrane multi-PDZ domain-containing proteins that are involved in regulation of intestinal NHE3. This study demonstrates that NHERF2 and NHERF3 have overlapping roles in NHE3 stimulation by LPA and inhibition by elevated Ca2+ and cGMP. These results are consistent with their role being as a NHERF3-NHERF2 heterodimer or via sequential NHERF-dependent signaling steps, and they begin to clarify a role for multiple NHERF proteins in NHE3 regulation.

Mixed Neurodevelopmental and Neurodegenerative Pathology in Nhe6-Null Mouse Model of Christianson Syndrome.

Christianson syndrome (CS) is an X-linked disorder resulting from loss-of-function mutations in SLC9A6, which encodes the endosomal Na+/H+ exchanger 6 (NHE6). Symptoms include early developmental delay, seizures, intellectual disability, nonverbal status, autistic features, postnatal microcephaly, and progressive ataxia. Neuronal development is impaired in CS, involving defects in neuronal arborization and synaptogenesis, likely underlying diminished brain growth postnatally. In addition to neurodevelopmental defects, some reports have supported neurodegenerative pathology in CS with age. The objective of this study was to determine the nature of progressive changes in the postnatal brain in Nhe6-null mice. We examined the trajectories of brain growth and atrophy in mutant mice from birth until very old age (2 yr). We report trajectories of volume changes in the mutant that likely reflect both brain undergrowth as well as tissue loss. Reductions in volume are first apparent at 2 mo, particularly in the cerebellum, which demonstrates progressive loss of Purkinje cells (PCs). We report PC loss in two distinct Nhe6-null mouse models. More widespread reductions in tissue volumes, namely, in the hippocampus, striatum, and cortex, become apparent after 2 mo, largely reflecting delays in growth with more limited tissue losses with aging. Also, we identify pronounced glial responses, particularly in major fiber tracts such as the corpus callosum, where the density of activated astrocytes and microglia are substantially increased. The prominence of the glial response in axonal tracts suggests a primary axonopathy. Importantly, therefore, our data support both neurodevelopmental and degenerative mechanisms in the pathobiology of CS.