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Christianson syndrome (CS) is an X-linked neurodevelopmental/neurological disorder characterized in males by the following core symptoms that include nonverbal status, intellectual disability, epilepsy, truncal ataxia, postnatal microcephaly and hyperkinesis. CS is caused by mutations in the SLC9A6 gene which encodes a multi-pass transmembrane sodi...
Citations
... Due to the random inactivation of either X chromosome, the X-linked gene Nexmif is predicted to be expressed in a mosaic manner in heterozygous female mice, leading to either normal expression or complete loss of NEXMIF in individual neurons. This type of mosaic expression has been observed in other haploinsufficient X-linked genes [51][52][53][54][55][56]. To determine the NEXMIF expression pattern in the brain, we used a NEXMIF specific antibody to immunolabel NEXMIF both ex vivo and in vivo. ...
Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a strong genetic basis. ASDs are commonly characterized by impairments in language, restrictive and repetitive behaviors, and deficits in social interactions. Although ASD is a highly heterogeneous disease with many different genes implicated in its etiology, many ASD-associated genes converge on common cellular defects, such as aberrant neuronal morphology and synapse dysregulation. Our previous work revealed that, in mice, complete loss of the ASD-associated X-linked gene NEXMIF results in a reduction in dendritic complexity, a decrease in spine and synapse density, altered synaptic transmission, and ASD-like behaviors. Interestingly, human females of NEXMIF haploinsufficiency have recently been reported to demonstrate autistic features; however, the cellular and molecular basis for this haploinsufficiency-caused ASD remains unclear. Here we report that in the brains of Nexmif± female mice, NEXMIF shows a mosaic pattern in its expression in neurons. Heterozygous female mice demonstrate behavioral impairments similar to those of knockout male mice. In the mosaic mixture of neurons from Nexmif± mice, cells that lack NEXMIF have impairments in dendritic arborization and spine development. Remarkably, the NEXMIF-expressing neurons from Nexmif± mice also demonstrate similar defects in dendritic growth and spine formation. These findings establish a novel mouse model of NEXMIF haploinsufficiency and provide new insights into the pathogenesis of NEXMIF-dependent ASD.
... Consistent with many X-linked disorders, skewed X-inactivation may play a role in the clinical presentation of SLC9A6 mutations in female carriers (6). Sikora et al. created a heterozygous SLC9A6 knockout female mouse that expressed the mutant SLC9A6 allele to follow the same neuroanatomic distribution and cell-specific patterns as hemizygous mutant males (21). The heterozygous knockout female mice developed progressive behavioral and neuropathological abnormalities that were similar to but milder than hemizygous male knockout mice. ...
Background
Intellectual disability, X-linked, syndromic, Christianson type (MRXSCH, OMIM: 300243)—known as Christianson syndrome (CS)—is characterized by microcephaly, epilepsy, ataxia, and absence of verbal language ability. CS is attributed to mutations in the solute carrier family 9 member A6 gene (SLC9A6).
Materials and methods
This study reports the case of a boy 1 year and 3 months of age who was diagnosed with CS in our department. Genetic etiology was determined by whole-exome sequencing, and a minigene splicing assay was used to verify whether the mutation affected splicing. A literature review of CS cases was conducted and the clinical and genetic features were summarized.
Results
The main clinical manifestations of CS include seizures, developmental regression, and exceptional facial features. Whole-exome sequencing revealed a de novo splice variant in intron 11 (c.1366 + 1G > C) of SLC9A6. The mutation produced two abnormal mRNA products (verified by a minigene splicing assay), resulting in the formation of truncated protein. A total of 95 CS cases were identified in the literature, with various symptoms, such as delayed intellectual development (95/95, 100.00%), epilepsy (87/88, 98.86%), and absent verbal language (75/83, 90.36%). At least 50 pathogenic variants of SLC9A6 have been identified, with the highest frequency observed in exon 12.
Conclusion
Our patient is the first case with the c.1366 + 1G > C variant of SLC9A6 in CS. The summary of known cases can serve as a reference for analyzing the mutation spectrum and pathogenesis of CS.
... In addition to its clear neurodevelopmental impacts, neurodegenerative features of NHE6 loss of function are increasingly appreciated. NHE6-null mice and rats exhibit progressive Purkinje cell loss, cortical thinning, and gliosis (Lee et al., 2021;Sikora et al., 2016;Stromme et al., 2011;Xu et al., 2017). NHE6-null rats show deposition of Ab and tau, as well as neuronal degeneration (Lee et al., 2021). ...
... Previous studies in NHE6-null mice and rats reported abnormal accumulation of GM2 ganglioside and unesterified cholesterol within late endosomes and lysosomes of neurons and reduced neuronal b-hexosaminidase activity (Lee et al., 2021;Sikora et al., 2016;Stromme et al., 2011); these phenotypes are typical of lysosomal storage disorders and suggest compromised lysosomal function in NHE6 KO neurons. In addition, we recently found that NHE6-null mouse neurons display defects in endosome maturation and endosome-lysosome fusion, which contribute to deficiencies in lysosome function . ...
Disruption of endolysosomal and autophagy-lysosomal systems is increasingly implicated in neurodegeneration. Sodium-proton exchanger 6 (NHE6) contributes to the maintenance of proper endosomal pH, and loss-of function mutations in the X-linked NHE6 lead to Christianson syndrome (CS) in males. Neurodegenerative features of CS are increasingly recognized, with postmortem and clinical data implicating a role for tau. We generated cortical neurons from NHE6 knockout (KO) and isogenic wild-type control human induced pluripotent stem cells. We report elevated phosphorylated and sarkosyl-insoluble tau in NHE6 KO neurons. We demonstrate that NHE6 KO leads to lysosomal and autophagy dysfunction involving reduced lysosomal number and protease activity, diminished autophagic flux, and p62 accumulation. Finally, we show that treatment with trehalose or rapamycin, two enhancers of autophagy-lysosomal function, each partially rescue this tau phenotype. We provide insight into the neurodegenerative processes underlying NHE6 loss of function and into the broader role of the endosome-lysosome-autophagy network in neurodegeneration.
... NHE6, an endosomal sodiumhydrogen exchanger known to be important in the regulation of endosomal pH, is encoded by Solute carrier family 9 member A6 (SLC9A6) (Yamashiro et al., 1984;Xinhan et al., 2011). Defects in the SLC9A6/NHE6 transporter have been linked to changes in somatosensory functions, according to a recent study (Sikora et al., 2016;Petitjean et al., 2020). Defects in the spinal cord, the nociceptor level, or supraspinal locations can cause sensory impairments. ...
Noncoding RNAs have been implicated in the pathophysiology of spinal cord injury (SCI), including cell death, glial scar formation, axonal collapse and demyelination, and inflammation. The evidence suggests that exercise therapy is just as effective as medical treatment in SCI. However, studies of competing endogenous RNA (ceRNA)-mediated regulation mechanisms in the therapy of SCI with exercise are rare. The focus of this research was to investigate the effect of exercise therapy on the expression levels of long noncoding RNA (lncRNA), microRNA (miRNA), and mRNA in rats with SCI. The RNA-seq technology has been used to examine the differentially expressed circRNAs (DECs), lncRNAs (DELs), miRNAs (DEMs), and genes (DEGs) between SCI and exercise therapy rats. The ceRNA network was established using interactions between miRNAs and mRNAs, as well as between miRNAs and lncRNAs/circRNAs. The Database for Annotation, Visualization, and Integrated Discovery was used to anticipate the underlying functions of mRNAs. Our current study identified 76 DELs, 33 DEMs, and 30 DEGs between groups of SCI rats and exercise therapy rats. Subsequently, these newly discovered ceRNA interaction axes could be important targets for the exercise treatment of SCI.
... Perhaps the most evident are deficits in locomotor control, as revealed by their relatively poor performance in rotarod and balance beam tasks when compared to age-and sex-matched wild-type (WT) mice (Stromme et al., 2011). These mice further exhibit hyperactivity and anxiety-type behaviours in open field paradigms (Stromme et al., 2011;Sikora et al., 2016), as well as impaired visuospatial learning (Sikora et al., 2016) and diminished sensitivity to pain and pressure stimuli (Kerner-Rossi et al., 2019;Petitjean et al., 2020). Interestingly, female heterozygous mice (Slc9a6 +/− ) show an intermediate phenotype compared to homozygous KO males (Slc9a6 -/Y ) and WT male and female mice (Sikora et al., 2016), mirroring clinical findings in female carriers of deleterious SLC9A6 alleles (Christianson et al., 1999;Pescosolido et al., 2019;Nan et al., 2022). ...
... Perhaps the most evident are deficits in locomotor control, as revealed by their relatively poor performance in rotarod and balance beam tasks when compared to age-and sex-matched wild-type (WT) mice (Stromme et al., 2011). These mice further exhibit hyperactivity and anxiety-type behaviours in open field paradigms (Stromme et al., 2011;Sikora et al., 2016), as well as impaired visuospatial learning (Sikora et al., 2016) and diminished sensitivity to pain and pressure stimuli (Kerner-Rossi et al., 2019;Petitjean et al., 2020). Interestingly, female heterozygous mice (Slc9a6 +/− ) show an intermediate phenotype compared to homozygous KO males (Slc9a6 -/Y ) and WT male and female mice (Sikora et al., 2016), mirroring clinical findings in female carriers of deleterious SLC9A6 alleles (Christianson et al., 1999;Pescosolido et al., 2019;Nan et al., 2022). ...
... These mice further exhibit hyperactivity and anxiety-type behaviours in open field paradigms (Stromme et al., 2011;Sikora et al., 2016), as well as impaired visuospatial learning (Sikora et al., 2016) and diminished sensitivity to pain and pressure stimuli (Kerner-Rossi et al., 2019;Petitjean et al., 2020). Interestingly, female heterozygous mice (Slc9a6 +/− ) show an intermediate phenotype compared to homozygous KO males (Slc9a6 -/Y ) and WT male and female mice (Sikora et al., 2016), mirroring clinical findings in female carriers of deleterious SLC9A6 alleles (Christianson et al., 1999;Pescosolido et al., 2019;Nan et al., 2022). These observations in heterozygotes are suggestive of a gene dosage effect of SLC9A6 mutations upon these phenotypes, as the extent (or skewing) of X-inactivation (Belmont, 1996;Plenge et al., 2002) of either the WT or mutant SLC9A6 allele in females likely accounts for the wide heterogeneity of their traits. ...
Endomembrane alkali cation (Na+, K+)/proton (H+) exchangers (eNHEs) are increasingly associated with neurological disorders. These eNHEs play integral roles in regulating the luminal pH, processing, and trafficking of cargo along the secretory (Golgi and post-Golgi vesicles) and endocytic (early, recycling, and late endosomes) pathways, essential regulatory processes vital for neuronal development and plasticity. Given the complex morphology and compartmentalization of multipolar neurons, the contribution of eNHEs in maintaining optimal pH homeostasis and cargo trafficking is especially significant during periods of structural and functional development and remodeling. While the importance of eNHEs has been demonstrated in a variety of non-neuronal cell types, their involvement in neuronal function is less well understood. In this review, we will discuss their emerging roles in excitatory synaptic function, particularly as it pertains to cellular learning and remodeling. We will also explore their connections to neurodevelopmental conditions, including intellectual disability, autism, and attention deficit hyperactivity disorders.
... Other groups have worked with Slc9a6 knockout mice as well as Slc9a6 mutant mice 37,55,56 to demonstrate that Slc9a6 loss of function causes the relevant defects, including disrupted endosomallysosomal function, neurodevelopmental and neurodegenerative pathology, impaired plasticity, and impoverished neuronal arborization 37,37,50,55,[57][58][59][60] . Further, Slc9a6 haploinsufficient mice develop PC, motor, and visuospatial abnormalities 61 . Other Slc9a6 rodent models are not reported as recapitulating all components of the human disease, such as seizures, cognitive deficits, or autistic behavior. ...
Background
The shaker rat carries a naturally occurring mutation leading to progressive ataxia characterized by Purkinje cell (PC) loss. We previously reported on fine-mapping the shaker locus to the long arm of the rat X chromosome. In this work, we sought to identify the mutated gene underlying the shaker phenotype and confirm its identity by functional complementation.
Methods
We fine-mapped the candidate region and analyzed cerebellar transcriptomes to identify deleterious variants. We generated an adeno-associated virus (AAV) targeting solute carrier family 9, member A6 ( Slc9a6) expression to PCs using a mouse L7-6 (L7) promoter, as well as a control green fluorescent protein (GFP)-expressing virus. We administered AAVs prior to the onset of PC degeneration through intracerebroventricular injection and evaluated the molecular, cellular, and motor phenotypes.
Results
We identified a XM_217630.9 ( Slc9a6 ):c.[191_195delinsA] variant in the Slc9a6 gene that segregated with disease. This mutation is predicted to generate a truncated sodium-hydrogen exchanger 6 (NHE6) protein, p.(Ala64Glufs*23). Administration of AAV9-PHP.eB expressing rat Slc9a6 prior to symptom onset reduced the shaker motor, molecular, and cellular phenotypes.
Interpretation
Slc9a6 is mutated in shaker and also in human Christianson syndrome, an epileptic encephalopathy. AAV-based gene therapy may be a viable therapeutic strategy for Christianson syndrome, and the shaker rat model may aid in therapeutic development.
... 11 NHE6-null mouse models exhibit reduced brain volume, increased gliosis, cerebellar degeneration and accumulation of lysosomal substrates in neurons. [13][14][15] Mouse models of neurodegenerative disorders, including of Alzheimer's disease, do not generally display deposition of endogenously expressed tau, [16][17][18] and mouse models of Christianson syndrome also do not exhibit tau deposition. 13 One study reports increases in amyloid-β by ELISA in the NHE6-null mouse brain, 19 although an increase in amyloid-β plaques are not reported in human post-mortem studies of Christianson syndrome. ...
... In terms of pathophysiological mechanisms, prior studies have demonstrated neuropathological hallmarks of lysosomal dysfunction, such as accumulation of GM2 ganglioside in NHE6-null mouse brains. 14,15 To investigate GM2 ganglioside accumulation in the cerebellum in the NHE6-null rat, the cerebellar sections from wild-type and NHE6-null rats at 2 months were co-stained with calbindin and GM2 (Fig. 3B). Strikingly, GM2 was detected in most Purkinje cell of NHE6-null rats, while it was not observed in those of wild-types. ...
... 7 However, this tau phenotype has not been recapitulated in the Christianson syndrome mouse models as reported previously, likely due to limitations of the mouse model to demonstrate tau pathology. [13][14][15] We also investigated amyloid-β accumulation in both NHE6-null rats and NHE6-null mice. Amyloid-β is produced by the cleavage of amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase in the amyloidogenic pathway. ...
Loss-of-function mutations in the X-linked endosomal Na+/H+ Exchanger 6 (NHE6) cause Christianson syndrome (CS) in males. CS involves endosome dysfunction leading to early cerebellar degeneration, as well as later-onset cortical and subcortical neurodegeneration, potentially including tau deposition as reported in postmortem studies. In addition, there is reported evidence of modulation of amyloid beta (Aβ) levels in experimental models wherein NHE6 expression was targeted. We have recently shown that loss of NHE6 causes defects in endosome maturation and trafficking underlying lysosome deficiency in primary mouse neurons in vitro. For in vivo studies, rat models may have an advantage over mouse models for the study of neurodegeneration, as rat brain can demonstrate robust deposition of endogenously-expressed Aβ and tau in certain pathological states. Mouse models generally do not show the accumulation of insoluble, endogenously-expressed (non-transgenic) tau or Aβ. Therefore, to study neurodegeneration in CS and the possibility of Aβ and tau pathology, we generated an NHE6-null rat model of CS using CRISPR-Cas9 genome-editing. Here, we present the sequence of pathogenic events in neurodegenerating NHE6-null male rat brains across the lifespan. NHE6-null rats demonstrate an early and rapid loss of Purkinje cells in the cerebellum, as well as a more protracted neurodegenerative course in the cerebrum. In both the cerebellum and cerebrum, lysosome deficiency is an early pathogenic event, preceding autophagic dysfunction. Microglial and astrocyte activation also occur early. In the hippocampus and cortex, lysosome defects precede loss of pyramidal cells. Importantly, we subsequently observe biochemical and in situ evidence of both Aβ and tau aggregation in the aged NHE6-null hippocampus and cortex (but not in the cerebellum). Tau deposition is widely distributed, including cortical and subcortical distributions. Interestingly, we observe tau deposition in both neurons and glia, as has been reported in CS postmortem studies previously. In summary, this experimental model is among very few examples of a genetically modified animal that exhibits neurodegeneration with deposition of endogenously-expressed Aβ and tau. This NHE6-null rat will serve as a new robust model for CS. Furthermore, these studies provide evidence for linkages between endo-lysosome dysfunction and neurodegeneration involving protein aggregations, including Aβ and tau. Therefore these studies may provide insight into mechanisms of more common neurodegenerative disorders, including Alzheimer’s Disease and related dementias.
... Abnormalities in these primary endosome processes may secondarily lead to defects in lysosome function. Neuropathological features seen in lysosome disorders have been previously reported in the CS mouse model (Stromme et al., 2011;Sikora et al., 2016); however, the molecular mechanisms underlying potential lysosome deficiency in CS are unknown. ...
... Neuropathological findings indicative of lysosome deficiency have been reported in a CS mouse model (Stromme et al., 2011;Sikora et al., 2016). These in vivo results are important as they strengthen the significance of our mechanistic studies here, indicating that our studies are not strictly attributable to the in vitro setting. ...
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, β-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.
... A CS-like phenotype is also reiterated in Slc9a6/Nhe6 knockout (KO) male mice (Nhe6 −/Y ), including atrophy and degeneration of cortical, hippocampal and especially cerebellar Purkinje neurons that parallel deficits in visuospatial memory and motor dexterity, and increased mortality prior to weaning (32)(33)(34). Likewise, heterozygous Nhe6 KO female mice (Nhe6 −/+ ) display a comparable, albeit milder, range of symptoms (35). ...
... Previous studies have revealed that genetic disruption of NHE6 expression causes degeneration of a subset of neurons within the cortex, hippocampus and especially the cerebellum which exhibits extensive Purkinje cell loss with age (32,34,35). Neurons in other brain regions, despite the loss of NHE6 expression, do not appear as severely affected. ...
... Less severe neuronal loss and gliosis have been documented in other brain regions, such as the cerebral cortex and hippocampus (6). Similar degeneration of the cerebellum and mild undergrowth and atrophy of the hippocampus, striatum, and cortex were also observed in Slc9a6 knockout mice (32,34,35). The variable penetrance and seemingly selective deterioration of certain neuronal populations upon loss of NHE6 function, despite its broad expression throughout the CNS, is not well understood but may reflect the nature of the mutation as well as other ill-defined genetic-modifier effects or compensatory mechanisms. ...
Genetic screening has identified several 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, 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 wild-type 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 also triggered apoptosis. These findings broaden our understanding of the molecular dysfunctions of distinct NHE6 variants associated with Christianson syndrome.
... The wide phenotypic spectrum reported in girls and women who are carriers of NHE6 mutations is consistent with other X-linked conditions wherein the tissues of female carriers are likely mosaic for gene expression (with a variable proportion of cells expressing from the mutant gene locus) based on random X-chromosome inactivation. Indeed, female Nhe6-heterozygous mice exhibit similar, yet less severe, neurological and motor phenotypes as Nhe6-null male mice [11]. As part of an international CS registry, we enrolled the largest cohort of female carriers (20 participants) across multiple CS pedigrees (n = 9). ...
... DNA was isolated from either blood or saliva from all female carriers. Heterozygosity for an NHE6 mutation known to cause CS in a related male was confirmed in participating females by sequencing all coding exons (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) and exon/intron junctions, as previously described [2]. Some pedigree features have been changed to assure anonymity. ...
... The most common pattern across individual female carriers was impairment in all three domains (39%). Interestingly, visuospatial memory deficits have also been reported in female Nhe6-heterozygous mice using a hippocampus-dependent object placement test [11]. Therefore, visuospatial abilities may represent a core domain affected by NHE6 dysfunction across species. ...
Mutations in NHE6 (also termed SLC9A6) cause the X-linked neurological disorder Christianson syndrome (CS) in males. The purpose of this study was to examine the phenotypic spectrum of female carriers of NHE6 mutations. Twenty female carriers from 9 pedigrees were enrolled, ranging from approximately age 2 to 65. A subset of female carriers was assessed using standardized neuropsychological measures. Also, the association of NHE6 expression with markers of brain age was evaluated using 740 participants in the Religious Orders Study (ROS) and Rush Memory and Aging Project (MAP). A majority, but not all, female carriers demonstrated a deficit in at least one neurocognitive domain (85%). A recognizable neuropsychological profile emerged, revealing impairments in visuospatial function, attention, and executive function. Common neuropsychiatric diagnoses included: intellectual disability/developmental delay (20%), learning difficulties (31%), speech/language delays (30%), and attention-deficit/hyperactivity disorder (20%). Notable neurological diagnoses in aging CS female carriers include corticobasal degeneration and atypical parkinsonism. In postmortem brains from the ROS/MAP dataset of normal and pathological aging, decreased NHE6 expression was correlated with greater tau deposition. Our study provides an examination of the phenotypic range in female carriers of NHE6 mutations. The findings indicate that NHE6-related disease in females represents a new neurogenetic condition.
