A mutation in the microtubule-associated protein tau in pallido-nigro-luysian degeneration

Materials and methods.

The patient was the youngest of four offspring of Japanese parents (figure 1). His father had parkinsonism and died in his forties and his paternal aunt died of psychosis and dementia. His older sister had parkinsonism with dementia and died in her forties. At age 41, his movements became slower, and hand tremor at rest was noticed. He developed difficulties in walking and had a mask-like depressive face for the next few years. At age 45, he was admitted to a hospital with several symptoms, including bradykinesia with rigidity and tremor, aggressiveness, psychosis with hallucinations and delusions, diminished spontaneous speech, and impaired memory. Levodopa reduced bradykinesia with rigidity to some extent, but at times it induced severe dyskinesia in his neck and upper extremities. His parkinsonian features, especially bradykinesia and rigidity, and dementia became significantly worse over the next 3 years. Neurologic examination revealed severe dementia, palilalia, vertical gaze palsy, blepharospasm, severe akinesia with rigid spasticity and tremor, nuchal stiffness, increased deep tendon reflexes with Babinski reflex and frontal lobe release signs, and urinary incontinence. He died of respiratory arrest at age 49.

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Figure 1. Pedigree of the family having a tau codon 279 mutation. An arrowhead indicates the proband of this family. Persons known to have dementia are indicated by black symbols. Children and grandchildren in more recent generations at risk for dementia are not included in the figure. Slash = deceased; squares = males; circles = females; diamonds = sex not declared.

Fourteen blocks were taken from the right hemisphere of the brain, fixed in 10% formalin, and embedded in paraffin. Eight-micrometer–thick sections were cut, stained with hematoxylin-eosin, Luxol fast blue-cresyl violet, modified Bielschowsky, and Gallyas-Braak stainings, and examined by light microscopy. In addition, immunohistochemical analysis was performed on some of these sections for tau protein. The primary antibody used in this study was a monoclonal antibody specific for nonphosphorylated Ser/Thr 189-207 (Tau-1 diluted at 1:200; Boehringer-Mannheim, Mannheim, Germany). Negative control samples were provided by omitting the primary or secondary antibody.

Genomic DNA was extracted from frozen temporal cortex. The target tau gene was amplified by PCR using primers derived from intronic sequences and sequenced as described previously.1 In addition, when a mutation was identified, we screened 100 controls and 50 patients with FTD who fulfilled the clinical criteria described previously7 by restriction fragment length polymorphism (RFLP).

An exon-10 fragment containing 33 bp and 51 bp of flanking intron sequences was generated by PCR. The PCR product was digested with XhoI and BamHI and inserted into vector pSPL3 (GIBCO-BRL, Life Technologies, Inc., Rockville, MD). Nucleotide changes in tau sequence were performed by PCR mutagenesis. All constructs were sequenced before transfection. For exon trapping, the exon-trapping system of Life Technologies was used according to manufacturer’s instructions.

Results.

Complete sequencing of the tau gene of the patient revealed a mutation (T to G) in one allele at nucleotide 837 in exon 10, which is predicted to cause an asparagine-to-lysine missense substitution at codon 279 (N279K, numbered from the 441-amino acid isoform of human brain tau). The substitution of T to G abolishes a restriction site for VspI (figure 2A). The PCR-RFLP analysis confirmed the heterozygote substitution at nucleotide 837 of the tau gene. The same substitution was not found in 100 healthy control subjects or in 50 patients with sporadic FTD. This mutation is the same as the one seen in the PPND family.5 The substitution at codon 279 is located in the inter-repeat region between the first and second tubulin-binding repeats (R1 and R2, respectively) of tau (figure 2B). The inter-repeat region between R1 and R2 of tau shares homology with at least two other MAPs, MAP2 and MAP4, in humans (figure 2C). In addition, the inter-repeat region between R1 and R2 is highly conserved between species. Asparagine (N) at codon 279 of human tau is conserved in bovine and murine tau, in murine MAP2, and in bovine and human MAP4 (figure 2C). The highly conserved nature of this site suggests that amino acid 279 is functionally significant.

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Figure 2. (A) VspI restriction enzyme analysis of the codon 279 (AAT to AAG) mutation. The amplification primers are forward: 5′- GCG TGT CAC TCA TCC TTT TT -3′, and reverse: 5′- AAT AAT TCA AGC CAC AGC AC -3′. The normal PCR product (N) was 175 base pairs (bp), which was cleaved to 126 and 49 bp fragments after VspI (GIBCO-BRL; Gaithersburg, MD) digestion. The patient (P) was heterozygous for a T-to-G transition that abolishes the VspI restriction site. When the amplified mutant fragment was digested overnight with VspI and resolved on a 2% NuSieve 3:1 agarose gel (FMC), a 175-bp band was left. (B) Amino-acid sequence (upper row) of the repeated domain of tau and nucleotide sequence (lower row) of the inter-repeated region. The first tubulin-binding repeat (R1) and the second tubulin-binding repeat (R2) are underlined. An arrow shows the T-to-G substitution in the inter-repeat region that is predicted to cause an asparagine-to-lysine missense substitution at codon 279 (N279K). (C) Homology between the first inter-repeated region of tau of different microtubule-associated proteins of human and other species. The asterisk indicates codon 279.

Discussion.

Recently, Goode and Feinstein8 found that the inter-repeat region between R1 and R2 can also bind to microtubules via an 8-amino acid–long sequence (274-KVQIINKK-281), where the N279K site is located. However, this mutation may not act at the protein level but rather may cause disease by altering splicing of tau exon 10. At the DNA sequence level, the 279 mutation occurs in a potential exon splicing enhancer (ESE) sequence that controls alternative splicing at exon 10. The 279 mutation is a T to G change that creates a GAR repeat motif (where R is a purine) that promotes incorporation of alternative spliced exons. This proposed mechanism is consistent with RNA splicing assays using the vector pSPL3, where the 279 mutation increases exon 10 incorporation relative to the normal tau exon 10 sequence (figure 3). Also, other studies reported the excess soluble four-repeat tau in brains from subjects with the 279 mutation.5

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Figure 3. Effect of the N279K mutation on splicing. (A) Autoradiograph of reverse transcription-PCR products from splicing assay. Exon 10-containing and exon 10-lacking transcripts yield 261-bp and 354-bp fragments, respectively. Products were resolved on 5% acrylamide gels. (B) Quantitation of exon 10-containing and exon 10-lacking splicing. Each bar represents the mean of three separate transfection experiments, and error bars are the standard deviations. *p < 0.001 versus normal tau.

The proband’s brain weighed 1,175 grams, exhibiting severe cortical atrophy; shrinkage of basal ganglia; marked dilatation of the lateral ventricles; discoloration of the striatum, globus pallidus, and luysian body; and prominent depigmentation of the substantia nigra and locus ceruleus. Gyral atrophy was especially evident in the frontal and anterior temporal lobes. The medial temporal lobe appeared moderately atrophic. Microscopic examination revealed moderate to severe neuronal loss, neuropil vacuolation of the superficial layers of the cerebral cortex, and variable astrocytic gliosis in both gray and white matter (figure 4). A large number of tau-positive ballooned neurons (figure 4H) and argyrophilic and tau-positive neuronal inclusion bodies, dystrophic neurites, and neuropil threads (figure 4, A through G) were seen especially in the frontal and anterior temporal cortices, but no Lewy bodies or Pick bodies were detected anywhere the brain. The amygdala and entorhinal cortex showed moderate neuronal loss and gliosis, whereas these changes were less prominent in the hippocampus. Although a few Alzheimer-type neurofibrillary tangles were seen, no extracellular amyloid deposits were detected even in the hippocampus. Loss of motor neurons and many inclusions within the remaining neurons were seen in the motor cortex and the brainstem motor nuclei such as the oculomotor nucleus. In addition to cortical lesions, neuronal loss and gliosis were remarkable in some subcortical structures, including the globus pallidus, nucleus basalis of Meynert (nbM), luysian body, substantia nigra, striatum, thalamus, and pedunculopontine tegmentum. Many degenerating neurons contained globose tangles in these subcortical lesions. Both cortical and subcortical lesions were found to contain conspicuous glial inclusions known as glial fibrillary tangles, which were also argyrophilic and tau-positive within oligodendrocytes (figure 4, B, C, I, and J).

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Figure 4. Tau pathology in a pallido-nigro-luysian degeneration (PNLD) brain. Numerous neuronal and glial inclusion bodies were found in PNLD. The cortex of the inferior frontal gyrus depicted numerous argyrophilic inclusions within neurons (thick arrow) in the gray matter (A) and within oligodendrocytes or astrocytes (thin arrows) in the gray (A) and white matter (B). Note many dystrophic neurites and neuropil threads (arrowheads) in gray matter and white matter as well as neuronal or glial tangles (arrows). A large number of abnormal argyrophilic structures were also visible in the atrophic globus pallidus (C) and amygdala (D). Some degenerating neurons contained reticular as well as globose argyrophilic inclusions (arrows in C and D). In the nucleus basalis of Meynert, there were many globose tangles, dystrophic neurites, and neuropil threads, all of which were argyrophilic (E) and immunolabeled for tau protein (F). Argyrophilic oligodendroglial fibrillary tangles (arrows) and interfascicular threads (arrowheads) were abundant in the internal capsule (G), and they were also immunolabeled with anti-tau antibodies (H). A–E and G, Gallyas-Braak silver staining; F and H, tau immunohistochemistry. Bar = 50 mm in all figures.

These pathologic findings indicate that our patient had tauopathy and shared pathologic features with PPND,9 a family classified as FTDP-17. The neuropathology of PNLD was distinct from the dominant pathologies of other FTDP-17 cases reported,10 as PNLD exhibited prominent changes in the motor cortex, motor nuclei of the brainstem, amygdala, and entorhinal cortex, with lesser alterations in the frontal and temporal cortices. The fact that the same amino acid change has been observed in two different families with very similar phenotypes but from different racial groups supports the hypothesis that 279K is a pathogenic mutation.