BACKGROUND: Christianson 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.
METHODS: Here, 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.
RESULTS: In 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.
CONCLUSIONS: These 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.