de novo MAPT mutation G335A causes severe brain atrophy, 3R and 4R PHF-tau pathology and early onset frontotemporal dementia

Main text Tauopathies are neurodegenerative disorders characterized by hyperphosphorylated microtubule-associated protein tau (MAPT) forming protein aggregates. Over sixty dominantly inherited mutations in MAPT have been reported. These mutations cause frontotemporal lobar degeneration with tau-immunoreactive inclusions (FTLD-tau) whose symptom onset and survival are highly heterogeneous [4]. We describe here the genetic, clinical, neuropathological and biochemical characterization of a case of de novo MAPTG335A mutation in a male patient. Symptoms of poor organisation skills, attention and concentration difficulties appeared when the proband was 14 years old. A few years later, he developed anxiety, panic attacks and time obsession. Firstly, he was considered to be suffering from a schizophrenic disorder. Since behavioral and phasic disturbances progressivly appeared, he was referred to a department of neurology at the age of 24. By this time, MRI showed a frontotemporal atrophy and SPECT demonstrated an hypofixation in the same regions. The clinical evolution was marked by a progressive decline in cognitive functions, particularly in speech output, semantic memory and in executive functions, an hyperorality, an emotional blunting and a buccofacial apraxia with swallowing disturbance necessiting an institutionalisation in an appropiate facility when he was 25. He never developed major extrapyramidal features, except a dystonia of upper left arm and a slight upper arms rigidity probably of iatrogenic origin (i.e. neuroleptic drugs). Sequencing of MAPT gene revealed a novel heterozygous mutation G335A due to G→C transition at the second base position in codon 335 in exon 12 (NM_ 005910.5: c.1004G > C p.Gly335Ala). The mutation was not found in his parents, thus strongly arguing for a de novo mutation. The proband died at 34, 20 years after the onset of symptoms. The proband’s brain was removed at autopsy, collected and stored in the brain bank of the LHNN (BB190052). One brain hemisphere was fixed in 10% buffered formalin. The other hemisphere was cut into coronal sections and kept at − 80 °C. A small piece of frontal cortex was fixed in 4% glutaraldehyde for transmission electron microscopy. The macroscopic examination showed a severe atrophy of the frontal and anterior temporal lobes (Fig. 1a) with a whole wet brain weight of 928 g. A marked dilatation of the frontal horn of the lateral ventricule was present (Fig. 1b). Neuropathological analysis was done on paraffin-embedded sections. In all regions examined, there were neurodegenerative changes consistent with a tauopathy (Table 1). Possible neuropathological

executive functions, an hyperorality, an emotional blunting and a buccofacial apraxia with swallowing disturbance necessiting an institutionalisation in an appropiate facility when he was 25. He never developed major extrapyramidal features, except a dystonia of upper left arm and a slight upper arms rigidity probably of iatrogenic origin (i.e. neuroleptic drugs).
Sequencing of MAPT gene revealed a novel heterozygous mutation G335A due to G → C transition at the second base position in codon 335 in exon 12 (NM_ 005910.5: c.1004G > C p.Gly335Ala). The mutation was not found in his parents, thus strongly arguing for a de novo mutation. The proband died at 34, 20 years after the onset of symptoms. The proband's brain was removed at autopsy, collected and stored in the brain bank of the LHNN (BB190052). One brain hemisphere was fixed in 10% buffered formalin. The other hemisphere was cut into coronal sections and kept at − 80°C. A small piece of frontal cortex was fixed in 4% glutaraldehyde for transmission electron microscopy.
The macroscopic examination showed a severe atrophy of the frontal and anterior temporal lobes (Fig. 1a) with a whole wet brain weight of 928 g. A marked dilatation of the frontal horn of the lateral ventricule was present (Fig. 1b). Neuropathological analysis was done on paraffin-embedded sections. In all regions examined, there were neurodegenerative changes consistent with a tauopathy (Table 1). Possible neuropathological Fig. 1 de novo mutation of MAPT-G335A led to severe brain atrophy with glial and neuronal tau pathology constituted of 3R and 4R tau. a Macroscopic pathology of FTLD-tau with MAPT-G335A mutation. The fixed left cerebral hemisphere showed a severe circumscribed atrophy of frontal lobe and anterior temporal lobe (with sparing of posterior superior gyrus). Motor and sensory gyri and occipital lobe were relatively spared. b Coronal sections of fixed brain. The frontal horn of the lateral ventricle was markedly dilated. c H&E reveals a ballooned neuron (right) and microvacuolation. d-e Immunostaining with AT8 (Thermo Scientific) (pS202/T205 tau) revealed numerous NFT in the pyramidal layer of the hippocampal CA1-2 sector (d). Occasionally there were astrocytic plaque-like appearance of tau-positive deposits in the striatum, thalamus and temporal cortex (e). f B19 (total tau)-positive astrocyte, reminiscent of tufted astrocytes were observed in the thalamus, frontal cortex and temporal cortex. g Coiled bodies (arrowhead) were immunolabelled by RD4 (clone 1E1/A6, Millipore) (4R tau). h Coiled bodies (arrowhead) were Gallyas positive. i-k Pyramidal neurons of the hippocampal CA2-3 sector immunostained for anti-tau B19 (i), RD4 (4R tau) (j) and Gallyas staining (k). Arrows show intraneuronal 4R tau positive globular and elongated inclusions (d, i-k). l-n NFT were immunostained with RD3 (clone 8E16/C11, Millipore, l) (3R tau) and RD4 (4R tau) (m). n Gallyas staining in CA2 sector shows a classical NFT (right) and Gallyas-positive intraneuronal threads (left). o-p Transmission electron microscopy showing a cytoplasmic aggregate of filaments in the frontal cortex. At higher magnification (p) of the inset in o, both straight filaments and PHF were observed. o 12,000x; p 30,000x. q Sarkosyl insoluble tau from the frontal cortex of the proband (MAPT-G335A) immunoblotted with PHF1, RD3, RD4 and total tau (A0024, Dako) antibodies. Sarkosyl insoluble tau is constituted of hyperphosphorylated 3R and 4R tau. Scale bars, 5 cm for a-b; 20 μm for c-n; 500 nm for o and 30 nm for p comorbidity was excluded by immunohistochemistry for Aß, α-synuclein and TDP-43. Significant neuronal loss and microvacuolation were observed in the superior layers of frontal and temporal cortex, where some ballooned neurons were also observed (Fig. 1c). In the deep layers of frontal and temporal cortex, there were neurofibrillary tangles (NFT) with flame shaped or globular forms, pre-tangles, neuropil threads, grain-like neuropil threads that were immunolabelled for total tau (B19) [2] and phosphotau AT8. Some NFT were detected in the parietal cortex but not in occipital cortex. NFT were prominent in the hippocampal CA sector and in the subiculum (Fig. 1d). Some astrocytes contained granular or punctate tau-immunoreactive deposits reminiscent of astrocytic plaques and tufted astrocytes (Fig. 1e, f). Tauimunoreactive astrocytes were predominantly 4R tau positive (Supplementary figure 1a-c, online resource). There were numbers of tau-positive coiled-bodies and axons in the white matter and in the deep cortical layers. The coiled bodies were labelled by 4R tau antibody and by Gallyas staining (Fig. 1g-h). Neuronal loss and NFT were prominent in substantia nigra. In the brainstem, tau positive lesions were detected in the pontine nuclei and in the olivary nuclei of the medulla. Some pyramidal neurons in hippocampal CA contained globular and elongated tau inclusions positive for 4R tau and Gallyas (Fig. 1d, i-k). NFT were detected by anti-3R and 4R tau antibodies (Fig. 1l, m). Some neurons had Gallyaspositive intraneuronal linear thread-like structures (Fig.  1n). GFAP positive gliosis was remarkable in the superior layers of the frontal cortex but less in the temporal cortex. A significant gliosis was observed in the sub-ependymal zone, especially at the level of the striatum and under the pia of the brainstem.
The ultrastructural aspect of the tau inclusions was analysed on ultrathin sections by transmission electron microscopy (Fig. 1o). Fibrillar inclusions were composed of both straight and paired helical filament (PHF) (Fig. 1p).
The sarkosyl fractionation method was used to enrich the insoluble tau from frozen grey matter of the frontal cortex [1] (Fig. 1q). By immunobloting, the sarkosyl insoluble tau extracted from the proband showed three major bands of 60, 64 and 68 kDa and was constituted of both 3R and 4R tau isoforms. This is the first report of the G335A mutation in exon 12 of MAPT that caused a strikingly early onset disease at 14 years old. Previously two MAPT mutations on the same amino acid have been reported to cause early onset frontotemporal dementia: the age of onset was 22 years old for MAPT-G335V [7] and 25.4 years old on average for MAPT-G335S [10]. Compared to other FTLD-tau-MAPT cases with an average age at onset of 49 years [4,8], mutations at MAPT-G335 led to early onset and severe neuronal and glial tau pathology ultrastructurally composed of straight filaments and PHF in MAPT-G335A (this study) and in MAPT-G335S [10]. The MAPT-G335S mutation was associated to neuronal tau pathology (tau positive neurons, NFT, neuropil threads) and glial tau pathology (coiled bodies, tau positive astrocytes), similarly to what we observed here in the MAPT-G335A mutation. Neuronal inclusions in the present MAPT-G335A were labelled with both anti-3R tau and anti-4R tau antibodies, but 3R tau positive Pick bodies were not detected, in contrast Table 1 Semi-quantitative assessment and regional distribution of tau pathology on brain sections immunolabelled with AT8. with other MAPT mutations such as G272V [3] and L266V [5]. The immunobloting pattern of sarkosylinsoluble tau in MAPT-G335A is similar to Alzheimer's disease [9]; the presence of both 3R and 4R tau isoforms in the insoluble fraction was also observed in MAPT-G389R [6] and MAPT-L266V [5]. This MAPT-G335A mutation changes the third among the four invariant PGGG motif in the microtubule-binding region to PGGA, and is predicted to affect the binding of tau to microtubules. Indeed, G335V and G335S mutations were reported to strongly affect this binding [7,10], a molecular effect that might lead to increased level of free tau and the assembly of tau into filaments.
Additional file 1: Figure S1. Tau positive astrocytes reminiscent of tufted astrocytes were predominantly detected by anti-4R tau RD4 antibody in the temporal cortex of MAPT-G335A brain. 4R tau isoform (green) is expressed in an astrocyte immunolabelled by the astrocyte marker GFAP (red). The mouse monoclonal anti-4R tau RD4 antibody (clone 1E1/ A6, Millipore) and the rabbit polyclonal anti-GFAP antibody (Sigma G9269) were detected as previously described [1].