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Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome.

ABSTRACT: Genetic screening has identified numerous variants of the endosomal solute carrier family 9 member A6 (SLC9A6)/(Na⁺,K⁺)/H⁺ exchanger 6 (NHE6) gene that cause Christianson syndrome, a debilitating X-linked developmental disorder associated with a range of neurological, somatic, and behavioral symptoms. Many of these variants cause complete loss of NHE6 expression, but how subtler missense substitutions or nonsense mutations that partially truncate its C-terminal cytoplasmic regulatory domain impair NHE6 activity and endosomal function are poorly understood. Here, we describe the molecular and cellular consequences of six unique mutations located in the N-terminal cytoplasmic segment (A9S), the membrane ion translocation domain (L188P and G383D), and the C-terminal regulatory domain (E547*, R568Q, and W570*) of human NHE6 that purportedly cause disease. Using a heterologous NHE6-deficient cell expression system, we show that the biochemical, catalytic, and cellular properties of the A9S and R568Q variants were largely indistinguishable from those of the WT transporter, which obscured their disease significance. By contrast, the L188P, G383D, E547*, and W570* mutants exhibited variable deficiencies in biosynthetic post-translational maturation, membrane sorting, pH homeostasis in recycling endosomes, and cargo trafficking, and they also triggered apoptosis. These findings broaden our understanding of the molecular dysfunctions of distinct NHE6 variants associated with Christianson syndrome.

SUBMITTER: Ilie A

PROVIDER: S-EPMC7242699 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome.

Ilie Alina A Boucher Annie A Park Jaeok J Berghuis Albert Marinus AM McKinney R Anne RA Orlowski John J

The Journal of biological chemistry 20200410 20

Genetic screening has identified numerous variants of the endosomal solute carrier family 9 member A6 (SLC9A6)/(Na+,K+)/H+ exchanger 6 (NHE6) gene that cause Christianson syndrome, a debilitating X-linked developmental disorder associated with a range of neurological, somatic, and behavioral symptoms. Many of these variants cause complete loss of NHE6 expression, but how subtler missense substitutions or nonsense mutations that partially truncate it ...[more]

PMID: 32277048

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Project description:BackgroundChristianson Syndrome, a recently identified X-linked neurodevelopmental disorder, is caused by mutations in the human gene SLC9A6 encoding the recycling endosomal alkali cation/proton exchanger NHE6. The patients have pronounced limitations in cognitive ability, motor skills and adaptive behaviour. However, the mechanistic basis for this disorder is poorly understood as few of the more than 20 mutations identified thus far have been studied in detail.MethodsHere, we examined the molecular and cellular consequences of a 6 base-pair deletion of amino acids Glu(287) and Ser(288) (∆ES) in the predicted seventh transmembrane helix of human NHE6 expressed in established cell lines (CHO/AP-1, HeLa and neuroblastoma SH-SY5Y) and primary cultures of mouse hippocampal neurons by measuring levels of protein expression, stability, membrane trafficking, endosomal function and cell viability.ResultsIn the cell lines, immunoblot analyses showed that the nascent mutant protein was properly synthesized and assembled as a homodimer, but its oligosaccharide maturation and half-life were markedly reduced compared to wild-type (WT) and correlated with enhanced ubiquitination leading to both proteasomal and lysosomal degradation. Despite this instability, a measurable fraction of the transporter was correctly sorted to the plasma membrane. However, the rates of clathrin-mediated endocytosis of the ∆ES mutant as well as uptake of companion vesicular cargo, such as the ligand-bound transferrin receptor, were significantly reduced and correlated with excessive endosomal acidification. Notably, ectopic expression of ∆ES but not WT induced apoptosis when examined in AP-1 cells. Similarly, in transfected primary cultures of mouse hippocampal neurons, membrane trafficking of the ∆ES mutant was impaired and elicited marked reductions in total dendritic length, area and arborization, and triggered apoptotic cell death.ConclusionsThese results suggest that loss-of-function mutations in NHE6 disrupt recycling endosomal function and trafficking of cargo which ultimately leads to neuronal degeneration and cell death in Christianson Syndrome.

Project description:Christianson syndrome (CS) is an X-linked neurodevelopmental and neurological disorder characterized in males by core symptoms that include non-verbal status, intellectual disability, epilepsy, truncal ataxia, postnatal microcephaly and hyperkinesis. CS is caused by mutations in the SLC9A6 gene, which encodes a multipass transmembrane sodium (potassium)-hydrogen exchanger 6 (NHE6) protein, functional in early recycling endosomes. The extent and variability of the CS phenotype in female heterozygotes, who presumably express the wild-type and mutant SLC9A6 alleles mosaically as a result of X-chromosome inactivation (XCI), have not yet been systematically characterized. Slc9a6 knockout mice (Slc9a6 KO) were generated by insertion of the bacterial lacZ/β-galactosidase (β-Gal) reporter into exon 6 of the X-linked gene. Mutant Slc9a6 KO male mice have been shown to develop late endosomal/lysosomal dysfunction associated with glycolipid accumulation in selected neuronal populations and patterned degeneration of Purkinje cells (PCs). In heterozygous female Slc9a6 KO mice, β-Gal serves as a transcriptional/XCI reporter and thus facilitates testing of effects of mosaic expression of the mutant allele on penetrance of the abnormal phenotype. Using β-Gal, we demonstrated mosaic expression of the mutant Slc9a6 allele and mosaically distributed lysosomal glycolipid accumulation and PC pathology in the brains of heterozygous Slc9a6 KO female mice. At the behavioral level, we showed that heterozygous female mice suffer from visuospatial memory and motor coordination deficits similar to but less severe than those observed in X-chromosome hemizygous mutant males. Our studies in heterozygous Slc9a6 KO female mice provide important clues for understanding the likely phenotypic range of Christianson syndrome among females heterozygous for SLC9A6 mutations and might improve diagnostic practice and genetic counseling by helping to characterize this presumably underappreciated patient/carrier group.

Project description:Mutations in solute carrier family 9 isoform 6 on chromosome Xq26.3 encoding sodium-hydrogen exchanger 6, a protein mainly expressed in early and recycling endosomes are known to cause a complex and slowly progressive degenerative human neurological disease. Three resulting phenotypes have so far been reported: an X-linked Angelman syndrome-like condition, Christianson syndrome and corticobasal degeneration with tau deposition, with each characterized by severe intellectual disability, epilepsy, autistic behaviour and ataxia. Hypothesizing that a sodium-hydrogen exchanger 6 deficiency would most likely disrupt the endosomal-lysosomal system of neurons, we examined Slc9a6 knockout mice with tissue staining and related techniques commonly used to study lysosomal storage disorders. As a result, we found that sodium-hydrogen exchanger 6 depletion leads to abnormal accumulation of GM2 ganglioside and unesterified cholesterol within late endosomes and lysosomes of neurons in selective brain regions, most notably the basolateral nuclei of the amygdala, the CA3 and CA4 regions and dentate gyrus of the hippocampus and some areas of cerebral cortex. In these select neuronal populations, histochemical staining for ?-hexosaminidase activity, a lysosomal enzyme involved in the degradation of GM2 ganglioside, was undetectable. Neuroaxonal dystrophy similar to that observed in lysosomal disease was observed in the cerebellum and was accompanied by a marked and progressive loss of Purkinje cells, particularly in those lacking the expression of Zebrin II. On behavioural testing, Slc9a6 knockout mice displayed a discrete clinical phenotype attributable to motor hyperactivity and cerebellar dysfunction. Importantly, these findings show that sodium-hydrogen exchanger 6 loss of function in the Slc9a6-targeted mouse model leads to compromise of endosomal-lysosomal function similar to lysosomal disease and to conspicuous neuronal abnormalities in specific brain regions, which in concert could provide a unified explanation for the cellular and clinical phenotypes in humans with SLC9A6 mutations.

Project description:BackgroundChristianson syndrome (CS) is caused by mutations in SLC9A6 and is characterized by global developmental delay, epilepsy, hyperkinesis, ataxia, microcephaly, and behavioral disorder. However, the molecular mechanism by which these SLC9A6 mutations cause CS in humans is not entirely understood, and there is no objective method to determine the pathogenicity of single SLC9A6 variants.MethodsTrio-based whole exome sequencing (WES) was carried out on two individuals with suspicion of CS. qRT-PCR, western blot analysis, filipin staining, lysosomal enzymatic assays, and electron microscopy examination, using EBV-LCLs established from the two patients, were performed.ResultsTrio-based WES identified a hemizygous SLC9A6 c.1560dupT, p.T521Yfs*23 variant in proband 1 and a hemizygous SLC9A6 c.608delA, p.H203Lfs*10 variant in proband 2. Both children exhibited typical phenotypes associated with CS. Expression analysis in EBV-LCLs derived from the two patients showed a significant decrease in mRNA levels and no detectable normal NHE6 protein. EBV-LCLs showed a statistically significant increase in unesterified cholesterol in patient 1, but only non-significant increase in patient 2 when stained with filipin. Activities of lysosomal enzymes (β-hexosaminidase A, β-hexosaminidase A + B, β-galactosidase, galactocerebrosidase, arylsulfatase A) of EBV-LCLs did not significantly differ between the two patients and six controls. Importantly, by electron microscopy we detected an accumulation of lamellated membrane structures, deformed mitochondria, and lipid droplets in the patients' EBV-LCLs.ConclusionsThe SLC9A6 p.T521Yfs*23 and p.H203Lfs*10 variants in our patients result in loss of NHE6. Alterations of mitochondria and lipid metabolism may play a role in the pathogenesis of CS. Moreover, the combination of filipin staining with electron microscopy examination of patient lymphoblastoid cells can serve as a useful complementary diagnostic method for CS.

Dataset Information

Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome.

Publications

Assorted dysfunctions of endosomal alkali cation/proton exchanger <i>SLC9A6</i> variants linked to Christianson syndrome.

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