A Christianson syndrome-linked deletion mutation (∆287ES288) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

Abstract

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 Glu287 and Ser288 (∆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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0129-9) contains supplementary material, which is available to authorized users.

Novel biomarkers to assess in utero effects of maternal opioid use: First steps toward understanding short‐ and long‐term neurodevelopmental sequelae

“…[49][50][51] In animals, maternal preconceptual opioid exposure is associated with altered sex-specific behavioral outcomes in both the first and second generations. 56 Although we do not wish to over interpret our results, we will have not yet been validated in the literature also show involvement in the pathogenesis of neurodevelopmental disorders (SLC9A6, 84,85 PPP1R15B 86 are associated with intellectual disability 87 ). 56 Although we do not wish to over interpret our results, we will have not yet been validated in the literature also show involvement in the pathogenesis of neurodevelopmental disorders (SLC9A6, 84,85 PPP1R15B 86 are associated with intellectual disability 87 ).…”

“…have not yet been validated in the literature also show involvement in the pathogenesis of neurodevelopmental disorders (SLC9A6, 84,85 PPP1R15B 86 are associated with intellectual disability 87 ). Upregulated miRs of interest in the buprenorphine-exposed group include miR 10b-5p, miR 10a-5p, miR 183-5p and miR 182-5p and alter the expression of molecules known to modulate synaptic plasticity that also contribute to the etiology of psychiatric diseases.…”

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

“…Thus in CS, it has been postulated that defects in NHE6 will compromise endosomal pH homeostasis and cargo trafficking in many tissues, but especially the CNS, leading to pleiotropic pathophysiological disturbances. This is supported by studies of mouse hippocampal neurons where disruption of NHE6 expression resulted in overacidification of early and recycling endosomes and diminished signalling from the tropomyosin or tyrosine receptor kinase B (TrkB) (33) and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (53,54) that correlated with reduced neurite outgrowth and branching, synapse density and maturation, and circuit strength.…”

“…To empirically visualize and evaluate the functional significance of the individual variants, we engineered them into the WT human NHE6 cDNA that was fused at its Cterminus to either enhanced green fluorescent protein (GFP), monomeric Cherry fluorescent protein (ChFP) or influenza hemagglutinin (HA)-epitope (NHE6GFP, NHE6ChFP, or NHE6HA respectively). We previously showed that the molecular and cellular properties of these chimeric constructs were indistinguishable from unmodified NHE6 when examined in AP-1 cells, a Chinese hamster ovary-derived cell line lacking detectable levels of endogenous NHE6 and therefore serves as a useful cell model system to study this transporter (53).…”

“…More than 80 genetic variants of NHE6 have been identified thus far (also see ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and DECIPHER (https://decipher.sanger.ac.uk/)), many of which are frameshift and nonsense mutations or mRNA splicing defects in the ion translocation domain (amino acids that are predicted to result in total loss of NHE6 protein. However, the consequences on endosomal and cellular function of other CSlinked NHE6 mutations that may not lead to a complete loss of protein expression or function have been less well documented (53,(55)(56)(57). These include missense substitutions, small inframe deletions, or nonsense mutations in the Cterminal regulatory domain that may generate protein products with altered functional properties (i.e., dominant-negative or constitutively-active) which, alongside other genetic factors, could contribute to reported variations in disease severity of CS patients (15).…”