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Extensive and progressive loss of Purkinje cells in the mouse cerebellum. (A-C) Immunoperoxidase staining for calbindin (brown) with Nissl counterstain (blue) revealed normal cerebellar Purkinje cells in a 19-day-old (p19) Slc9a6 À /Y animal. The arrows in C indicate axonal spheroids. (D) Purkinje cell degeneration, particularly noticeable in lobules I/II and III at p57. (E) Purkinje cell loss and axonal spheroids (encircled) at p57. (F) Purkinje cell that has lost all dendritic branches (ML = molecular layer; PCL = Purkinje cell layer; GCL = granular cell layer). (G) Purkinje cell degeneration is extensive with relative sparing of lobules IX and X at p153. (H) Purkinje cells in the process of losing dendritic branches. (I) Magnified view of insert in G.
Source publication
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 co...
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... extensive and progressive loss of Purkinje cells observed in the cerebellum of Slc9a6 mutant mice is illustrated in Fig. 6. Another striking feature was the presence of Purkinje cell axonal spheroids ( Fig. 6C and E). The spheroids appeared similar to those observed in a number of lysosomal disorders, including Niemann-Pick dis- ease type C ( Walkley et al., 2010). The loss of Purkinje cells was not random but instead occurred in a regular pattern of ...
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... extensive and progressive loss of Purkinje cells observed in the cerebellum of Slc9a6 mutant mice is illustrated in Fig. 6. Another striking feature was the presence of Purkinje cell axonal spheroids ( Fig. 6C and E). The spheroids appeared similar to those observed in a number of lysosomal disorders, including Niemann-Pick dis- ease type C ( Walkley et al., 2010). The loss of Purkinje cells was not random but instead occurred in a regular pattern of parasag- ittal stripes in the cerebellar vermis (Figs 7, 8A and B) and hemi- spheres (Fig. 8C). ...
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... such as the amygdala and CA3 and CA4 regions of the hippocampus. In the basolateral nuclei of the amygdala, NHE6 expression was not especially con- spicuous, yet this was the site of massive GM2 ganglioside accu- mulation. Strong NHE6 tissue expression in the Purkinje cell layer of the cerebellum (Fig. 1F and G) coincided with Purkinje cell loss (Fig. 6), but without abnormal GM2 ganglioside accumulation or related evidence of endosomal or lysosomal ...
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... have found a number of pathological defects in the cerebel- lum of Slc9a6-targeted mice. Purkinje cell axons exhibited axonal spheroids ( Fig. 6C and E) that are swellings on the axon, typically seen in Niemann-Pick disease type C and many other lysosomal diseases ( Walkley et al., 2010). The loss of Purkinje cells occurred in parasagittal stripes (Fig. 7). This is not the first demonstration of patterned cell loss in the cerebellum. For example, in the spon- taneous mutants ...
Citations
... Further and importantly, by constructing gene co-expression networks using the weighted gene-co-expression network analysis (WGCNA) approach, we identified dysregulated lysosome-related genes, significantly co-expressed in NHE6 null lines. There is some prior evidence of lysosome mechanisms implicated in CS pathogenesis (Stromme, Dobrenis et al. 2011, Fernandez, Bah et al. 2022. Therefore, in this study, we present a comprehensive and wellcontrolled analysis of the transcriptome architecture of NHE6 mutations in a new, human haploid NHE6 null cell line, which potentially provides insight into disease mechanisms in CS. ...
... In this network, we also noted upregulation of multiple lysosome disease genes, genes for related neurologic disease and other endolysosome genes: TPP1, the causation for neuronal ceroid lipofuscinosis (NCL) type 2; CLN3, the causation for NCL type 3; GDAP2, associated with spinocerebellar ataxia; CD63, RAB7A, LAMP1, M6PR, or PLA2G15. These associations with lysosome mechanisms and lysosome disease is in strong concordance with prior biological studies in CS mouse studies both in vitro ) and in vivo (Stromme, Dobrenis et al. 2011). Therefore, these studies here, in a simple cell model, now provide valuable data supporting the idea that disruption of NHE6 leads to primary and fundamental defects in lysosome function, which is consistent with the argument from prior investigators who have argued that CS may be a lysosomal disorder or on a continuum with these disorders. ...
Christianson Syndrome (CS) is an X-linked disorder resulting from loss-of-function (LoF) mutations in SLC9A6 encoding the endosomal Na+/H+ exchanger 6 (NHE6). CS presents with developmental delay, seizures, intellectual disability, nonverbal status, postnatal microcephaly, and ataxia. To define transcriptome signatures of NHE6 LoF, we conducted in-depth RNA-sequencing (RNA-seq) analysis on a haploid NHE6 null cell model. CRIPSR/Cas9 genome editing introduced multiple LoF mutations into SLC9A6 in the near haploid human cell line Hap1. Isogenic, paired parental controls were also studied. NHE6 mutant cell lines were confirmed to have intra-endosomal over-acidification as was seen in other NHE6 null cells. RNA-seq analysis was performed by two widely used pipelines: HISAT2-StringTie-DEseq2 and STAR-HTseq-DEseq2. We identified 1056 differentially expressed genes in mutant NHE6 lines, including genes associated with neurodevelopment, synapse function, voltage-dependent calcium channels, and neuronal signaling. Weighted Gene Co-Expression Network Analysis was then applied and identified a critical module enriched for genes governing lysosome function. By identifying significantly changed gene expression that is associated with lysosomal mechanisms in NHE6-null cells, our analyses suggest that loss of NHE6 function may converge on mechanisms implicated in lysosome-related neurologic disease. Further, this haploid cell model will serve as an important tool for translational science in CS.
... The presence of the LacZ-Neo at this site added a stop codon and a polyadenylation termination signal, which halted the transcription of the target gene resulting in the deficient mice. The tails clippings from the homozygous Nhe6 deficient male and female mice and the litters were used for genotyping as per the procedure outlined in ref. 65. The mice were housed, fed a standard diet and maintained at the animal facility. ...
Cellular sodium ion (Na⁺) homeostasis is integral to organism physiology. Our current understanding of Na⁺ homeostasis is largely limited to Na⁺ transport at the plasma membrane. Organelles may also contribute to Na⁺ homeostasis; however, the direction of Na⁺ flow across organelle membranes is unknown because organellar Na⁺ cannot be imaged. Here we report a pH-independent, organelle-targetable, ratiometric probe that reports lumenal Na⁺. It is a DNA nanodevice containing a Na⁺-sensitive fluorophore, a reference dye and an organelle-targeting domain. By measuring Na⁺ at single endosome resolution in mammalian cells and Caenorhabditiselegans, we discovered that lumenal Na⁺ levels in each stage of the endolysosomal pathway exceed cytosolic levels and decrease as endosomes mature. Further, we find that lysosomal Na⁺ levels in nematodes are modulated by the Na⁺/H⁺ exchanger NHX-5 in response to salt stress. The ability to image subcellular Na⁺ will unveil mechanisms of Na⁺ homeostasis at an increased level of cellular detail.
... encodes the endosomal Na + /H + exchanger 6 (NHE6). 2,9,10 It is expressed broadly, with highest expression in the central nervous system, especially in the hippocampus, cortex, and cerebellum, [11][12][13] which likely explains the marked neurological morbidity of CS. 12 NHE6 is predominantly localized to early and recycling endosomes. 14,15 It plays a role for the processing and trafficking of intracellular cargo, likely by regulating endosomal pH of early and recycling endosomes through exchanging luminal H + for Na + . ...
... 14,15 It plays a role for the processing and trafficking of intracellular cargo, likely by regulating endosomal pH of early and recycling endosomes through exchanging luminal H + for Na + . Moreover, NHE6 has been proposed to play roles in growth of dendritic spines and neuronal arborization, [11][12][13] indicating that NHE6 is critical to normal nervous system development and function. Indeed, deletion of SLC9A6 in mice results in endosomallysosomal dysfunction as evidenced by the intraneuronal accumulation of GM2 ganglioside and unesterified cholesterol, accompanied by a progressive loss of Purkinje cells. ...
... Indeed, deletion of SLC9A6 in mice results in endosomallysosomal dysfunction as evidenced by the intraneuronal accumulation of GM2 ganglioside and unesterified cholesterol, accompanied by a progressive loss of Purkinje cells. 11 Notably, β-hexosaminidase activity, which is involved in the lysosomal degradation of GM2 ganglioside, was almost undetectable in GM2-accumulating neurons. 11 Currently, the diagnosis of CS is based on genetic testing. ...
Background
Christianson 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.
Methods
Trio‐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.
Results
Trio‐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.
Conclusions
The 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.
... BPAN (WDR45) Pathogenic variants of WDR45, encoding for WIPI4 protein, may cause iron accumulation in the basal ganglia by impeding autophagy, 67 that may result in neuroinflammation and swelling of the substantia nigra.SLC9A6 encodes the endosomal Na+/H+ exchanger 6 and is involved in endosomal luminal pH and trafficking, synapse development, and plasticity.68 Findings in Slc9a6 knock-out mice were consistent with endosomal-lysosomal dysfunction.69 ATP6V1B2 encodes a subunit of the lysosomal transmembrane proton pump. ...
Background:
With advances in clinical genetic testing, associations between genetic neurodevelopmental disorders and parkinsonism are increasingly recognized. In this review, we aimed to provide a comprehensive overview of reports on parkinsonism in genetic neurodevelopmental disorders and summarize findings related to genetic diagnosis, clinical features and proposed disease mechanisms.
Methods:
A systematic literature review was conducted in PubMed and Embase on June 15, 2021. Search terms for parkinsonism and genetic neurodevelopmental disorders, using generic terms and the Human Phenotype Ontology, were combined. Study characteristics and descriptive data were extracted from the articles using a modified version of the Cochrane Consumers and Communication Review Group's data extraction template. The protocol was registered in PROSPERO (CRD42020191035).
Results:
The literature search yielded 208 reports for data-extraction, describing 69 genetic disorders in 422 patients. The five most reported from most to least frequent were: 22q11.2 deletion syndrome, beta-propeller protein-associated neurodegeneration, Down syndrome, cerebrotendinous xanthomatosis, and Rett syndrome. Notable findings were an almost equal male to female ratio, an early median age of motor onset (26 years old) and rigidity being more common than rest tremor. Results of dopaminergic imaging and response to antiparkinsonian medication often supported the neurodegenerative nature of parkinsonism. Moreover, neuropathology results showed neuronal loss in the majority of cases. Proposed disease mechanisms included aberrant mitochondrial function and disruptions in neurotransmitter metabolism, endosomal trafficking, and the autophagic-lysosomal and ubiquitin-proteasome system.
Conclusion:
Parkinsonism has been reported in many GNDs. Findings from this study may provide clues for further research and improve management of patients with GNDs and/or parkinsonism.
... Homozygous male Nhe6 KO (Slc9a6 -/Y ) mice exhibit several neurobehavioural abnormalities that resemble clinical phenotypes seen in CS patients. 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). ...
... 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). ...
... Indeed, Nhe6 KO mice do exhibit a pronounced increase in activated astrocytes and microglia within their gray matter (Xu et al., 2017;Kerner-Rossi et al., 2019). This observation may be a response to the heightened neurodegeneration detected in CS patients (Christianson et al., 1999;Gilfillan et al., 2008;Garbern et al., 2010;Schroer et al., 2010) and NHE6 KO mice (Stromme et al., 2011;Xu et al., 2017), but it could also be indicative of possible neuroinflammation which may not directly arise from its role in astrocytes. Interestingly, though not covered in this review, diminished NHE6 expression has also been detected in astrocytes associated with Alzheimer's disease, suggesting a broader role for NHE6 is neurodegenerative pathophysiology (Prasad and Rao, 2018a;Prasad and Rao, 2018b). ...
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.
... Our work led to the discovery of a novel PTV (p.A64Efs*23) in Slc9a6 in the shaker rat, making this the first rat model of CS. 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 . ...
... In terms of PC degeneration, reports of NHE6-null mice indicate a slower progression, perhaps with less severe PC loss. 59,72 Among the possible underlying mechanisms, one involves differential activity of NHE6 paralogs such as NHE9, which is also localized in recycling endosomes. Indeed, there exists a complex interplay between vesicular ATP-dependent proton pumps and the NHEs, and it is possible that any number of modifiers may yield important differences in endosomal pH and PC health. ...
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.
... Sanger sequencing was performed to validate the potentially pathogenic variations identified in the patient and his parents to determine the parental origin. The phenotypes of the proband and his parents were verified according to published articles (1,3,(7)(8)(9)(10)(11)(12)(13) and OMIM database (OMIM:300243). ...
... NHE6 abnormalities can also affect cell viability (14), possibly due to disruption of the balance of apoptosis as a result of endosomal dysfunction and attenuation of tyrosine receptor kinase B (TrkB) signaling (8), although the mechanism remains to be elucidated. SlC9A6 knockout mice presented an abnormal accumulation of GM2 ganglioside and unesterified cholesterol within late endosomes and lysosomes, with slow degeneration of neurons in the hippocampus, some areas of cerebral cortex and cerebellar Purkinje cells and a CS-like clinical phenotype (7). In addition to NHE6 loss-of-function mutations, which have been confirmed to be correlated to the pathophysiology of CS with SLC9A6 variants, NHE6 gain-of-function mutations have also been reported to be associated with impairment of optimal recycling endosomal function, accounting for the pathophysiology of CS (12). ...
... The correlation with CS has been well-established. CS follows a X-linked recessive inheritance pattern, with clinical features that overlap with those of AS in male patients presenting with moderate to severe global developmental delay, epilepsy, absent or impaired speech, truncal ataxia, ophthalmoplegia, acquired microcephaly, hyperkinesis, cerebellar atrophy and reduced life expectancy ( Table 1) (1,3,(7)(8)(9)(10)(11)(12)(13). Seizure onset usually occurs in late infancy. ...
We investigated the existence and potential pathogenicity of a SLC9A6 splicing variant in a Chinese boy with Christianson Syndrome (CS), which was reported for the first time in China. Trio whole-exome sequencing (WES) was performed in the proband and his parents. Multiple computer prediction tools were used to evaluate the pathogenicity of the variant, and reverse transcription-polymerase chain reaction (RT-PCR) analysis and cDNA sequencing were performed to verify the RNA splicing results. The patient presented with characteristic features of CS: global developmental delay, seizures, absent speech, truncal ataxia, microcephaly, ophthalmoplegia, smiling face and hyperkinesis with electrical status epilepticus during sleep (ESES) detected in an electroencephalogram (EEG). A SLC9A6 splicing variant was identified by WES and complete skipping of exon 10 was confirmed by RT-PCR. This resulted in altered gene function and was predicted to be pathogenic. ESES observed early in the disease course is considered to be a significant feature of CS with the SLC9A6 variant. Combined genetic analysis at both the DNA and RNA levels is necessary to confirm the pathogenicity of this variant and its role in the clinical diagnosis of CS.
... 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. ...
... Data from ageing Christianson syndrome patients and NHE6-null mice demonstrate mixed neurodevelopmental and neurodegenerative pathology. 5,8,13,14 To understand the trajectories of brain morphology changes in male NHE6-null rats, we measured the length of the anterior-posterior (A-P) axis of brains from wild-type and their NHE6-null littermates at 3, 9 and 12 months ( Fig. 2A and B). The A-P length of 3-month-old NHE6-null rats was shorter than wild-type even though the brain size of NHE6-null rats continued to increase (Fig. 2B). ...
... 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. ...
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. ...
... 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. In Stromme et al. (2011), NHE6-null mice exhibit features consistent with lysosomal storage diseases, such as pathologic accumulation of GM2 ganglioside and unesterified cholesterol in late endosomes and lysosomes that affect particular brain regions, including the hippocampus. In the current study, we directly measured lysosomal degradation of endocytosed cargo in vitro. ...
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.
... Cerebellar heterogeneity is also illustrated by the nonhomogeneous and nonrandom PC degeneration across the cerebellar cortex observed in a broad spectrum of cerebellar pathologies. These cerebellar pathologies, displayed by patients or rodent models, can result from genetic mutations, such as leaner (15), Harlequin (16), sticky (17), Slc9a6 −/− (18), and Cav2.1 −/− (19), as well as brain ischemia (20), viral infections (21), prion diseases (22), and alcohol abuse, such as alcohol-related cerebellar degeneration (23). As described by Sarna and Hawkes (24), not all patterned neurodegenerations are similar. ...
Significance
Neuronal subtypes are differentially affected by neuropathologies. For example, Purkinje cells, the principal neurons of the cerebellum, can be divided in subpopulations based on their sensitivity to pathological insult. However, the molecular mechanisms explaining why, among seemingly identical neurons, some will degenerate while others survive remain unknown. Here, we analyzed, in a disease model of cerebellar neurodegeneration, the metabolism of sphingolipids, complex lipids involved in cell apoptosis, and found that specific sphingolipids accumulate in the cerebellar region primarily affected by neurodegeneration. Preventing this accumulation by disrupting sphingolipid metabolism via genetic mutation caused a neuroprotective effect on subpopulations of Purkinje cells. Thus, our data indicate that sphingolipid metabolism is involved in the predisposition of neuronal subtypes to neurodegeneration.
