Neurobiology of Disease 121 (2019) 187–204 Contents lists available at ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi A potential gain-of-function variant of SLC9A6 leads to endosomal T alkalinization and neuronal atrophy associated with Christianson Syndrome Alina Iliea, Andy Y.L. Gaob, Annie Bouchera, Jaeok Parkc, Albert M. Berghuisc, ⁎ Mariëtte J.V. Hofferd, Yvonne Hilhorst-Hofsteed, R. Anne McKinneyb, John Orlowskia, a Department of Physiology, McGill University, Montreal, Canada b Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada c Department of Biochemistry, McGill University, Montreal, Canada d Department of Clinical Genetics, Leiden University Medical Center, Leiden, the Netherlands ARTICLE INFO ABSTRACT Keywords: Loss-of-function mutations in the recycling endosomal (Na+,K+)/H+ exchanger gene SLC9A6/NHE6 result in Christianson Syndrome overacidification and dysfunction of endosomal-lysosomal compartments, and cause a neurodevelopmental and X-linked intellectual disability degenerative form of X-linked intellectual disability called Christianson Syndrome (CS). However, knowledge of Alkali cation/proton exchangers –SLC9A6/ the disease heterogeneity of CS is limited. Here, we describe the clinical features and underlying molecular and NHE6 cellular mechanisms associated with a CS patient carrying a de novo missense variant (p.Gly218Arg; G218R) of a Endosomal pH homeostasis conserved residue in its ion translocation domain that results in a potential gain-of-function. The patient Membrane trafficking Neurodegeneration manifested several core symptoms typical of CS, including pronounced cognitive impairment, mutism, epilepsy, ataxia and microcephaly; however, deterioration of motor function often observed after the first decade of life in CS children with total loss of SLC9A6/NHE6 function was not evident. In transfected non-neuronal cells, com- plex glycosylation and half-life of the G218R were significantly decreased compared to the wild-type transporter. This correlated with elevated ubiquitination and partial proteasomal-mediated proteolysis of G218R. However, a major fraction was delivered to the plasma membrane and endocytic pathways. Compared to wild-type, G218R- containing endosomes were atypically alkaline and showed impaired uptake of recycling endosomal cargo. Moreover, instead of accumulating in recycling endosomes, G218R was redirected to multivesicular bodies/late endosomes and ejected extracellularly in exosomes rather than progressing to lysosomes for degradation. Attenuated acidification and trafficking of G218R-containing endosomes were also observed in transfected hippocampal neurons, and correlated with diminished dendritic branching and density of mature mushroom- shaped spines and increased appearance of filopodia-like protrusions. Collectively, these findings expand our understanding of the genetic diversity of CS and further elucidate a critical role for SLC9A6/NHE6 in fine-tuning recycling endosomal pH and cargo trafficking, processes crucial for the maintenance of neuronal polarity and mature synaptic structures. Abbreviations: AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; Arf6, ADP-ribosylation factor 6; ARNO, ADP-ribosylation factor nucleo- tide site opener; AP-1, Chinese hamster ovary (CHO) cells deficient in the+ Na /H+ exchanger 1 isoform; CANX, calnexin; ChFP, monomeric Cherry fluorescent protein; CNS, central nervous system; CS, Christianson syndrome; DMSO, dimethylsulfoxide; EV, extracellular vesicles; ER, endoplasmic reticulum; ERAD, en- doplasmic reticulum-associated degradation; ESCRT, endosomal sorting complex required for transport; FRIA, fluorescence ratiometric image analysis; G218R, mutation of amino acid Gly to Arg at position 218 in NHE6v1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HA, influenza virus hemagglutinin epitope; Hsp70, heat shock protein 70; Lamp1, lysosome-associated membrane protein 1; LE, late endosomes; LeuP, leupeptin/pepstatin; M.I.F, median intensity fluorescence; MVB, multivesicular bodies; OMIM, Online Mendelian Inheritance in Man; PCR, polymerase chain reaction; pHv, vesicular pH;PM, plasma membrane; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SLC9A6/NHE6, (sodium, potassium)/proton exchanger isoform 6; TCL, total cell lysate; Tf, transferrin; TfR, transferrin receptor; TGN, trans-Golgi network; TRKB, tropomyosin receptor kinase B; TSG 101, tumor susceptibility gene 101 protein; Ub, ubiquitin; WT, wild-type; XLID, X-linked intellectual disability. ⁎ Corresponding author at: Department of Physiology, McGill University, Bellini Life Sciences Bldg., Rm, 166 3649 Promenade Sir-William-Osler, Montreal H3G 0B1, Canada. E-mail address: [email protected] (J. Orlowski). https://doi.org/10.1016/j.nbd.2018.10.002 Received 19 July 2018; Received in revised form 18 September 2018; Accepted 3 October 2018 Available online 05 October 2018 0969-9961/ © 2018 Elsevier Inc. All rights reserved. A. Ilie et al. Neurobiology of Disease 121 (2019) 187–204 1. Introduction Arg) of the affected male that results in substitution of a highly con- served glycine at position 218 with an arginine in the transmembrane Christianson Syndrome (CS; OMIM 300243) is a rare but increas- domain of the longest splice-variant of NHE6 (i.e., NHE6v1). We in- ingly diagnosed neurodevelopmental and regressive form of X-linked vestigate the effects of this mutation on the biosynthetic processing, intellectual disability (XLID) first described in a multigenerational stability and membrane trafficking of NHE6 and its impact on en- South African family in 1999 (Christianson et al., 1999). The causative dosomal function and neuronal morphology. These results identify a gene, SLC9A6 (solute carrier family 9, member A6), was identified at novel molecular pathology for organellar pH-regulating transporters chromosomal position Xq26.3 in 2008 and encodes the (Na+,K+)/H+ and provides further insight into the pathogenesis of this important exchanger isoform 6 (also called NHE6)(Gilfillan et al., 2008). Since neurological syndrome. then, over 50 different mutations in NHE6 have been identified worldwide, many causing premature truncation of the protein in its N- 2. Materials and methods terminal transmembrane domain that likely results in total loss-of- function (Gilfillan et al., 2008; Pescosolido et al., 2014) (also see sup- 2.1. Antibodies and reagents plementary data Tables S1 and S2, and databases ClinVar (https:// www.ncbi.nlm.nih.gov/clinvar/) and DECIPHER (https://decipher. Mouse monoclonal anti-hemagglutinin (HA) antibody was purchased sanger.ac.uk/)). The CS population frequency is estimated to range from Covance Inc. (Berkeley, CA); rabbit polyclonal anti-HA, mouse from 1 in 16,000 to 100,000 (Pescosolido et al., 2014), although this monoclonal anti-GAPDH, rabbit polyclonal anti-Lamp1, and rabbit mono- may be an underestimate due to incomplete diagnoses. In addition to clonal anti-TSG101 were obtained from Abcam Inc. (Cambridge, MA); significant limitations in cognitive abilities and social/behavioral mouse monoclonal anti-Flag M2 antibody was from Sigma; rabbit poly- adaptive skills, all affected males lack speech despite noticeable audi- clonal anti-GFP antibody was from Life Technologies. Rabbit polyclonal tory perception, and most have seizures, microcephaly, ataxia, atrophy anti-calnexin and mouse monoclonal anti-Hsp70 antibodies were from and neuronal loss in brain regions including the cerebellum and brain Enzo Life Sciences, Inc. Mouse monoclonal anti-ubiquitin antibody (P4D1) stem, and reduced life expectancy. While most female carriers are was obtained from Santa Cruz Biotechnology. Rabbit polyclonal anti- asymptomatic, they may present with mild learning and behavioral cleaved caspase-3 (Asp175) (cCASP3) antibody was purchased from Cell difficulties (Christianson et al., 1999; Masurel-Paulet et al., 2016; Signaling Technology. The mEmerald GFP-tagged TGN46 was a gift from Sinajon et al., 2016). Deficits in NHE6 function may also extend to Michael Davidson (Addgene, mEmerald-TGNP-N-10 plasmid # 54279). other neurodevelopmental disorders, as significantly reduced NHE6 Rab4-GFP, Rab5-GFP, and Rab7-GFP were a kind gift from Dr. Terry gene expression has been described in postmortem cerebral cortex from Hébert (McGill University). Horseradish peroxidase-conjugated secondary patients with idiopathic autism (Schwede et al., 2013); further sug- IgG antibodies were purchased from Jackson ImmunoResearch gesting a critical role for this transporter in cognitive development. Laboratories (West Grove, PA). All Alexa Fluor® conjugated secondary However, the spectrum and frequency of clinical symptoms and geno- antibodies were purchased from Molecular Probes (Eugene, OR). Alpha- type-phenotype correlations remain to be documented. minimum essential medium (α-MEM), fetal bovine serum, penicillin/ NHE6 is expressed in most tissues (Fagerberg et al., 2014) but is streptomycin, and trypsin-EDTA were purchased from Wisent (Saint-Bruno, highest in the central nervous system (CNS), consistent with the pro- QC, Canada). The DMEM/F12 medium was from Corning. All other che- minent neuropathologic phenotype of CS. In non-neuronal cells, it is mical and reagents were obtained from BioShop Canada (Burlington, ON, abundant in recycling endosomes together with the transferrin re- Canada), Sigma or Fisher Scientific and were of the highest grade available.