A mutation affecting the sodium/proton exchanger, SLC9A6, causes mental retardation with tau deposition

Abstract

We have studied a family with severe mental retardation characterized by the virtual absence of speech, autism spectrum disorder, epilepsy, late-onset ataxia, weakness and dystonia. Post-mortem examination of two males revealed widespread neuronal loss, with the most striking finding being neuronal and glial tau deposition in a pattern reminiscent of corticobasal degeneration. Electron microscopic examination of isolated tau filaments demonstrated paired helical filaments and ribbon-like structures. Biochemical studies of tau demonstrated a preponderance of 4R tau isoforms. The phenotype was linked to Xq26.3, and further analysis identified an in-frame 9 base pair deletion in the solute carrier family 9, isoform A6 (SLC9A6 gene), which encodes sodium/hydrogen exchanger-6 localized to endosomal vesicles. Sodium/hydrogen exchanger-6 is thought to participate in the targeting of intracellular vesicles and may be involved in recycling synaptic vesicles. The striking tau deposition in our subjects reveals a probable interaction between sodium/proton exchangers and cytoskeletal elements involved in vesicular transport, and raises the possibility that abnormalities of vesicular targeting may play an important role in more common disorders such as Alzheimer's disease and autism spectrum disorders.

Figure 1

Pedigree of the family. Patient IV-8 is mildly retarded but has acquired good language skills. Patient V-3, by age 5 years, had acquired some receptive language ability, was able to operate a videotape player and other household appliances and had been diagnosed as mildly autistic, and is highlighted by the corner square. The propositus is indicated by an arrow. The subjects who were examined and from whom DNA samples were obtained are indicated by a dot to the lower left of the individual’s icon. Plus symbols indicate individuals who have the c.1012_1020del mutation in SLC9A6. Female heterozygotes are indicated by the central dot.

Figure 2

Representative MRI slices from Patient III-9. (A) Axial T₂-weighted image showing moderate ventricular enlargement and sulcal widening with normal appearance of white matter. (B) Axial T₂-weighted image at level of midbrain. In addition to the atrophied appearance of the brain, there is a left parieto-occipital lesion (arrowhead) with evidence of surrounding past haemorrhage (dark rim). (C) Gadolinium enhanced T₁-weighted slice showing enhancement of the left parieto-occipital lesion (arrowhead). (D) T₁ sagittal slice through the midline showing thin corpus callosum and moderate sulcal prominence and ventricular dilatation.

Figure 3

Histological and immunohistochemical analyses. (A) Cerebellar cortex from Patient III-9 showing severe loss of Purkinje cells and neuronal loss in the internal granular layer. Haematoxylin–eosin stain, scale bar = 300 µm. (B) Cerebral white matter from Patient III-11 stained with anti-tau showing diffuse abundant positive staining of white matter glial elements. Scale bar = 100 µm. (C) Tau-stained substantia nigra from Patient III-9 showing frequent tau-positive neuronal inclusions. Scale bar = 20 µm. (D) Tau immunostaining of the third cranial nerve nucleus from Patient III-11 shows abundant positive staining of the neuronal population. Scale bar = 200 µm. (E) Tau-positive astrocytic plaque (arrowhead) in the dorsal raphae nucleus in Patient III-9. Scale bar = 20 µm. (F) Bielschowsky silver stain of the dentate nucleus of the cerebellum in Patient III-11 showing frequent silver-positive neuronal inclusions. Scale bar = 50 µm.

Figure 4

(A) Isoform composition of sarkosyl-insoluble tau. Frozen tissue from frontal grey (Fg) and white (Fw) matter and basal ganglia (BG) from Case III-9 were used to extract sarkosyl-insoluble tau species. A portion of each sample was dephosphorylated by incubation with alkaline phosphatase (AP). The dephosphorylated (AP +) and non-dephosphorylated (AP−) samples plus a mixture of six recombinant human brain tau isoforms were resolved on 7.5% SDS–PAGE and immunoblotted with a mixture of monoclonal antibodies T14 and T46. (B–D) Ultrastructural analysis of isolated tau filaments revealing paired-helical filament-like structures (B and C) as well as ribbon-like structures (D).

Figure 5

(A) Solubility profile of tau proteins. Frontal grey (Fg) and white (Fw) matter from Case III-9 as well as basal ganglia (BG) from Case III-11 were used for sequential extraction of tau with buffers of increasing stringency. Proteins in each fraction were separated by SDS–PAGE and immublotted with a mixture of monoclonal antibodies T14 and T46. 1 = high-salt; 2 = 1% Triton-X100; 3 = radioimmunoprecipitation assay buffer; 4 = 2% SDS; 5 = 70% formic acid fractions. (B) Insoluble tau proteins in the 2% SDS fractions are hyperphosphorylated as shown by immunoblotting with phosphorylation-specific monoclonal antibodies PHF1, AT8 and 12E8.