The novel MAPT mutation K298E: mechanisms of mutant tau toxicity, brain pathology and tau expression in induced fibroblast-derived neurons

In 2009, a 67-year-old lady was referred to the neurology service for investigation of progressive gait difficulties and non-fluent aphasia. She had previously developed a tremor of her right hand at the age of 48, which had been non-troubling. At the age of 65, the tremor became more severe and also spread to involve the left hand such that she had to give up writing and also driving. At that time her MMSE score was 27/30 and a CT brain scan revealed bilateral cortical atrophy. SPECT imaging by [(123) I] FP-CIT (DaTscan) was normal. Over the next 2 years, her condition progressed and she suffered a decline in her balance and mobility with initially recurrent falls and then progressing such she could no longer stand or sit unaided. She also exhibited a decline in her speech with severe word finding difficulties. This was associated with marked anxiety and cognitive decline. She had now become dependent for all her daily living activities. During this time there had been no fluctuation in her condition and no history of visual hallucinations. Her past medical history included sick sinus syndrome and she had been fitted with a cardiac pacemaker. The family history was relevant as her father was diagnosed with Alzheimer’s disease in his 60 s and died at the age of 67. There were no other family members with dementia. She was a non-smoker and there was no history of alcohol excess.

Significant examination findings included: an unreactive facies and severe cognitive impairment, which prevented completion of a full MMSE. There was a paucity of speech and her speech was effortful. She was able to register three objects but could not recall any. She was poor at naming objects. She could perform a one-stage command but not three. She exhibited perseveration. She had prominent primitive reflexes including grasp reflexes and rooting response. In her limbs, she had a bilateral symmetrical myoclonic tremor (arms > legs), which was irregular and jerky. She had increased tone in all limbs with marked spasticity of her right arm. Her deep tendon reflexes were brisk (right > left) with down-going plantar responses.

Blood investigations, including treponemal serology and vasculitic screen, were unremarkable. A repeat CT brain scan showed progression of the cerebral atrophy. CSF analysis was normal. EEG revealed intermittent slow waves, mainly in the right hemisphere, suggesting cortical dysfunction. Genetic analysis of MAPT revealed a novel single nucleotide change from A>G (AAA to AAG) at codon 298 in exon 10 which resulted in a change from Lys 298 to Glu in one allele. The proband’s condition progressed and she died in 2010.

Microtubule assembly assays were performed with recombinant wild-type or mutant tau proteins carrying the K298E mutation or the previously characterized P301S mutation. Both P301S and K298E recombinant proteins showed a reduced ability to promote microtubule assembly compared to wild-type tau (Fig. 1), suggesting that the K298E mutation disrupts the ability of tau to interact with microtubules.

We next examined the effect of the K298E tau mutation on exon 10 alternative mRNA splicing in cultured cells. A minigene construct with A892>G mutation was engineered based on the previously described human tau minigene model [17]. Human NSCs were transiently transfected at day 20 of differentiation with mutant K298E or the wild-type tau minigene, as described [1]. Untransfected human NSCs expressed a strong 3R tau isoform and a very weak 4R tau band at the mRNA but not the protein levels as previously shown [15] (Fig. 2a, lane 1). When transfected with the empty vector or the wild-type tau minigene, the level of expression of 3R tau remained high. In contrast, transfection of the I10+3 mutation minigene (Fig. 2a, lane 4) or the K298E mutant minigene (Fig. 2a, lane 5) altered the ratio between 3R and 4R tau isoforms in favour of 4R tau, indicating that the two mutations strongly reduced the splicing out of exon 10. Co-transfection of the wild-type minigene and the K298E minigene still increased the amount of transcripts with exon 10, indicating that the K298E mutation had a dominant effect on the splicing of exon 10 (Fig. 2a, lane 6). Figure 2b shows a quantification of tau isoform mRNA levels in human NSCs in three different experiments for each line. The effect of the K298E on exon 10 splicing was confirmed by RT-PCR of RNA extracted from the patient’s brain. A clear increase of the 4R tau band was observed compared to the RT-PCR of RNA extracted from the cortex of a control subject (Fig. 2c).

The left half of the brain weighed 513 g. External examination and coronal slices showed severe atrophy of the frontal lobe, including the motor strip, and the anterior temporal lobe, while the parietal and posterior temporal lobes were less severely affected and there was good preservation of the occipital lobe. There was severe dilatation of the frontal and temporal horns of the lateral ventricle. The cortical ribbon was thin throughout the frontal lobe and in all three temporal gyri anteriorly with patchy thinning in the parietal lobe and in the second and third temporal gyri posteriorly. There was atrophy of the caudate nucleus and thalamus with preservation of the putamen, globus pallidus and subthalamic nucleus. The hippocampus and amygdala were severely atrophic. Both the substantia nigra and the locus coeruleus were pale. Medulla and cerebellum were unremarkable.

Immunohistochemistry showed widespread neuronal loss and gliosis with abundant deposition of hyperphosphorylated tau in neurons and glia. The semiquantitative assessment of these features in different brain regions is detailed in Table 2.

In addition to cortical thinning, gliosis and mild superficial spongiosis in the frontal, temporal and parietal cortices, a number of ballooned neurons were observed, they were particularly prominent in the cingulate gyrus (Fig. 3a–c). Neuronal loss was most severe in the frontal and parietal cortices, the caudate nucleus and in the CA3 and CA4 subregions of the hippocampus (Fig. 4a). Although neuronal loss was not marked in the dentate fascia of the hippocampus, there was dispersion of the granule cell layer (Fig. 4a). Poorly defined basophilic inclusions were evident in a proportion of neurons in haematoxylin- and eosin-stained sections (Fig. 3d). Tau immunohistochemistry revealed extensive neuronal and glial deposition of phosphorylated tau (AT8, epitope phosphoSer202 and phosphoThr205) (Figs. 3e–k, 4c–h). Neuronal inclusions of various types were observed: crescentic or ring-like perinuclear inclusions; diffuse cytoplasmic staining, sometimes also containing fine filaments; flame-shaped inclusions and globular inclusions. The former were particularly marked in the small neurons of the superficial cortical laminae (Fig. 3f–j). Astrocytic tau pathology was observed with morphological features similar to tufted astrocytes (Fig. 3m, o, q). In white matter there were frequent tau-immunoreactive oligodendroglial inclusions, numerous threads and dots (Fig. 3k). All of these structures were immunoreactive for 4R tau and astrocytic pathology was most clearly visible in this preparation (Figs. 3l, m, n; 4b). 3R tau was also detected in a proportion of inclusions, predominantly neuronal, using immunohistochemistry, but immunoreactivity was much more restricted than that of 4R tau (Fig. 3l). AT100-positive staining recognized a proportion of neuronal inclusion and threads. P62 immunohistochemistry highlighted abnormal astrocytes, some of the neuronal inclusions and a proportion of oligodendroglial inclusions in white matter (Fig. 3o). Ubiquitin immunohistochemistry revealed weak immunoreactive inclusions mostly with a morphology compatible with astrocytic inclusions. Gallyas silver staining demonstrated a proportion of neuronal, astrocytic and oligodendroglial inclusions, in addition to some of the thread pathology indicating fibrillar tau deposition (Fig. 3p–r). There was no TDP-43 or Aβ pathology in neocortical regions or the hippocampus. No α-synuclein pathology in the form of Lewy bodies, Lewy neurites or glial cytoplasmic inclusions was found in brainstem, limbic or cortical regions.

To measure the level of expression of tau isoforms, soluble tau was extracted with perchloric acid from the frontal cortex of a patient with the I10+3 mutation, normal adult human brain and the frontal cortex of the proband with the K298E mutation. All six tau isoforms were expressed in the normal, I10+3 and K298E tau mutant brains (Fig. 5a); however, in the K298E case the 4R tau isoforms 1N4R appeared increased and the 3R tau isoforms 1N3R decreased (Fig. 5a). A slight increase in the 0N4R tau isoform could also be observed in the K298E case. These data showed and confirmed that tau pathology in the frontal cortex had a high prevalence of 4R tau isoforms. Sarkosyl-insoluble tau extraction from the frontal cortex of the proband with the K298E MAPT mutation confirmed that 4R tau was the major component, although some 3R tau was also present (Fig. 5b). Sarkosyl-insoluble tau extracted from the brain of the I10+3 case, where filaments had been shown to contain 4R tau, is shown for comparison.

Direct conversion of human fibroblasts into neurons is of potential clinical utility and of interest for neurological disease modelling, as well as for cell therapeutics [3]. A skin biopsy was taken from the patient with the K298E mutation 2 h post-mortem and fibroblasts were derived. These were then expanded and converted into iNs, as previously described [24, 25]. As controls, human embryonic fibroblasts (hEFs) and human adult fibroblasts (hAFs) were converted into neurons following an identical protocol. Two weeks after transgene activation, induced neurons started to appear in both controls and K298E cells, but tau was not detected (data not shown). Thirty days after transgene activation, while iNs obtained from human embryonic fibroblasts (hEF-iN) expressed only the 3R tau isoform, iNs with the K298E mutation (iN-K298E) and iNs from hAF (hA-iN) expressed both 3R and 4R tau (Fig. 6a). Fifty-three days after transgene activation, the level of expression of both tau isoforms in the iN-K298E did not increase (Fig. 6b) and was comparable to the level of tau observed in hAF-iNs. However, hEF-iNs still expressed only 3R tau 53 days after transduction (Fig. 6b). The limited availability of fibroblasts and differentiated neurons did not allow for biochemical studies or more indepth analyses.