Qi et al.
Acta Neuropathologica Communications(2023) 11:74
https://doi.org/10.1186/s40478-023-01565-2

Acta Neuropathologica
Communications

Open Access

RESEARCH

Identical tau filaments in subacute sclerosing
panencephalitis and chronic traumatic
encephalopathy
Chao Qi1†, Masato Hasegawa2†, Masaki Takao3,4, Motoko Sakai5, Mayasuki Sasaki6, Masashi Mizutani3,
Akio Akagi7, Yasushi Iwasaki7, Hiroaki Miyahara7, Mari Yoshida7, Sjors H. W. Scheres1* and Michel Goedert1*   

Abstract
Subacute sclerosing panencephalitis (SSPE) occurs in some individuals after measles infection, following a symptomfree period of several years. It resembles chronic traumatic encephalopathy (CTE), which happens after repetitive head
impacts or exposure to blast waves, following a symptom-free period. As in CTE, the neurofibrillary changes of SSPE
are concentrated in superficial cortical layers. Here we used electron cryo-microscopy (cryo-EM) of tau filaments fromtwo cases of SSPE to show that the tau folds of SSPE and CTE are identical. Two types of filaments were each made of
two identical protofilaments with an extra density in the β-helix region. Like in CTE, the vast majority of tau filaments
were Type I, with a minority of Type II filaments. These findings suggest that the CTE tau fold can be caused by different environmental insults, which may be linked by inflammatory changes.
Keywords Tau, Subacute sclerosing panencephalitis, Chronic traumatic encephalopathy, Inflammation, Electron
cryo-microscopy†

Chao Qi and Masato Hasegawa are contributed equally to this work

*Correspondence:
Sjors H. W. Scheres
scheres@mrc-lmb.cam.ac.uk
Michel Goedert
mg@mrc-lmb.cam.ac.uk
1
Medical Research Council Laboratory of Molecular Biology, Cambridge,UK
2
Department of Brain and Neuroscience, Tokyo Metropolitan Instituteof Medical Science, Tokyo, Japan3
Department of Clinical Laboratory and Internal Medicine, National
Center of Neurology and Psychiatry, Tokyo, Japan
4
Department of Neurology and Brain Bank, Mihara Memorial Hospital,
Isesaki, Japan
5
Department of Neurology, National Hospital Organization Suzuka
National Hospital, Suzuka, Mie, Japan
6
Department of Child Neurology, Center of Neurology and Psychiatry,Tokyo, Japan
7
Department of Neuropathology, Institute for Medical Science of Aging,
Aichi Medical University, Nagakute, Aichi, Japan

Introduction
Subacute sclerosing panencephalitis (SSPE) is a fatal disorder of the central nervous system that occurs following infection with measles virus and manifests itself after
a symptom-free period of several years [1]. It occurs inapproximately 1 in 75,000 cases of measles [2]. The neuropathology of SSPE is characterized by severe nerve cellloss, demyelination, perivascular lymphocytic infiltrations and viral intranuclear inclusion bodies. By silver
staining, abundant neurofibrillary tangles are present in
cerebral cortex and other brain regions in a proportionof cases [3, 4]. They have been reported to be made ofpaired helical filaments like those from Alzheimer’s disease brains [5, 6] and stain for abnormally phosphorylated tau and ubiquitin [7]. Tangle-bearing cases of SSPE
have mostly a long disease duration [8] and in a recent
immunohistochemical study, tau pathology was found in
all cases of SSPE [9].

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Qi et al. Acta Neuropathologica Communications(2023) 11:74

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Tauopathy has been inferred to result from diffuse
brain inflammation triggered by infection with measles
virus and not from a direct effect of the virus [9]. Neurofibrillary tangles of SSPE stain with antibodies specificfor 3R and 4R tau, like the tau inclusions of primary agerelated tauopathy (PART), Alzheimer’s disease (AD) andchronic traumatic encephalopathy (CTE) [10]. Unlike
PART and AD, but like CTE, the neurofibrillary tangles
of SSPE are present in superficial cortical layers [9, 11].
We previously used electron cryo-microscopy (cryoEM) to determine the atomic structures of tau filaments from a number of neurodegenerative conditions,which has resulted in a structure-based classification oftauopathies [12]. We showed that the tau filament folds
of 3R + 4R tauopathies separate into two groups, the firstof which is formed by PART, AD, and familial British andDanish dementias (FBD and FDD), and the second by
CTE. The neurofibrillary pathology associated with somecases of Gerstmann-Sträussler-Scheinker disease (GSS)
also belongs to the first group [13]. We now report thatthe structures of tau filaments from two cases of SSPE
are identical to those of CTE. This suggests that the CTE
tau fold can form in response to different environmental
insults, which may be linked by inflammatory changes.

aged 41. The brain was severly atrophic with a weight of
735 g. Pathological examination revealed the presence ofmarked brain atrophy with nerve cell loss and gliosis incerebral cortex, basal ganglia, thalamus and hippocampus, which were associated with severe white matter
atrophy [9]. Tau immunoreactive NFTs showed a broad
distribution, in particular in superficial layers II and III of
the cerebral cortex, in oculomotor nuclei and in the locus
coeruleus.

Materials and methods

Immunolabelling and histology

Clinical history and neuropathology

Immunogold negative-stain electron microscopy andimmunoblotting were carried out as described [15]. For
immunoelectron microscopy, the samples were applied
onto collodion membrane-applied mesh, blocked with
0.3% gelatin, incubated with AT8 (1:100, Innogenetics
90,206) for 1 h at 37 °C, followed by a 1 h incubation at
37 °C with 10 nm gold-labelled secondary antibody (1:50)
and staining with 2% phosphotungstate. For immunoblotting, samples were run on 5–20% gradient gels (FujiFilm). Proteins were then transferred to a polyvinylidene
difluoride membrane and incubated with the following
primary antibodies overnight at room temperature: Tau
N (1:1000, Cosmobio TIP-TAU-P-03), AT8 (1:500), RD3(1:500, Millipore 05-803), RD4 (1:500, Millipore 05-804),
anti-4R (1:1000, BioLegend MMS-5020), Tau354-369(1:1000, Millipore ABN2178-100UL) and T46 (1:1000,
Thermo Fisher Scientific 13-6400). Following washing in
PBS, the membranes were incubated with biotinylated
anti-mouse or anti-rabbit secondary antibody (Vector, 1:500) for 1 h at room temperature, followed by a
30 min incubation with avidin–biotin complex and colour development using NiCl-enhanced diaminobenzidine as substrate. Histology and immunohistochemistrywere carried out as described [16]. Brain sections were
8 µm thick and were counterstained with haematoxylin.
Primary antibodies were: RD3 (1:1000); anti-4R (1:1000);
AT8 (1:300); anti-measles virus fusion protein antibody

We determined the cryo-EM structures of tau filamentsfrom the frontal cortex of two individuals with SSPE.
There was no history of head injury. Case 1 was a male
who developed measles when 1.5 years old; at age 8, hedeveloped a speech disturbance, as well as eating andwalking difficulties. He was diagnosed with SSPE basedon clinical presentation, a history of measles infection
and characteristic electro-encephalogram and cerebrospinal fluid abnormalities. Despite intensive antiviraltreatment, his condition worsened progressively and hedied aged 42, after having been on mechanical ventilation for 11 years. The brain was severely atrophic witha weight of 439 g. Neuronal rarefaction was extensive
in cerebrum, brainstem and cerebellum and there was asevere loss of myelinated nerve fibres. Tau-immunoreactive NFTs showed a wide distribution and were present
in layers II and III of the cerebral cortex. The clinicopathological characteristics of SSPE case 2 have been
described [case 4 in [9]]. Briefly, this was a male who
developed measles at the age of 0.8 year; at age 22, hedeveloped convulsions, myoclonus and parkinsonism. He
was diagnosed with SSPE based on the neurological findings, a history of measles infection, periodic synchronous
discharge on electro-encephalogram and cerebrospinal
fluid abnormalities. Intensive antiviral treatment failed
to improve his symptoms, he became bedridden and died

Extraction of tau filaments

Sarkosyl-insoluble material was extracted from the frontal cortex of cases 1 and 2 of SSPE, as described [14], withminor modifications. Briefly, tissues were homogenizedwith a Polytron in 40 vol (w/v) extraction buffer consisting of 10 mM Tris–HCl, pH 7.4, 0.8 M NaCl, 10% sucroseand 1 mM EGTA. Homogenates were brought to 2%
sarkosyl and incubated for 30 min at 37 °C. Following a
10 min centrifugation at 27,000 g, the supernatants werespun at 257,000 g for 30 min. Pellets were resuspended in
2 ml extraction buffer containing 1% sarkosyl and centrifuged at 166,000 g for 20 min. The resulting pellets were
resuspended in 100 µl phosphate-buffered saline (PBS)
and used for subsequent analyses.

Qi et al. Acta Neuropathologica Communications(2023) 11:74

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Fig. 1 Frontal cortex from SSPE case 1: Immunohistochemical characterisation of tau inclusions and inflammatory changes. a RD3 (specific for
3R tau)-immunoreactive nerve cells and neuropil threads. b RD4 (specific for 4R tau)-immunoreactive nerve cells and neuropil threads. c AT8(specific for pS202 and T205 tau)-immunoreactive nerve cells and neuropil threads. d Antibody against measles virus shows neuronal staining.
e, Iba-1-immunoreactive microglial cells. f CD3-immunoreactive lymphocytes. Many tau inclusions were found in layers II and III. Scale bars: a–e,
50 µm; f, 100 µm.

(1:1000, Funakoshi bs-0886R); Iba-1 (1:2000, 019-19741);
CD3 (1:1000, Novocastra NCL-1-CD3-565).
Electron cryo‑microscopy: sample preparation and datacollection

Extracted tau filaments were centrifuged at 3000 g for
1 min and applied to UltrAuFoil cryo-EM grids [17],which were glow-discharged with an Edwards (S150B)
sputter coater at 30 mA for 1 min. Aliquots of 3 µl wereapplied to the glow-discharged grids, blotted with filter paper and plunge-frozen into liquid ethane using a

Vitrobot Mark IV (Thermo Fisher Scientific) at 100%
humidity and 4 °C. Cryo-EM images were collected on a
Titan Krios electron microscope (Thermo Fisher Scientific) operated at 300 kV and equipped with a Falcon-4
direct electron detector. Images were recorded during 6 s
exposures in EER (electron event representation) format[18] with a total dose of 40 electrons per ­A2 and a pixel
size of 0.824 Å.

Qi et al. Acta Neuropathologica Communications(2023) 11:74

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density using Coot [27]. Model refinements were performed using Servalcat [28] and Refmac5 [29, 30]. Models were validated with MolProbity [31]. Figures wereprepared with ChimeraX [32] and Pymol [33]. Further
details of data acquisition and image processing are given
in Additional file 1: Table S1 and Figure S1.

Fig. 2 Immunolabelling and immunoblotting of tau filaments from
SSPE. a, b Immunoelectron microscopy of filaments from SSPE cases
1 (a) and 2 (b) using anti-tau antibody AT8. c Immunoblotting ofsarkosyl-insoluble fractions using anti-tau antibodies: Tau N; AT8; RD3;
Anti-4R; Tau354; T46. Lanes: 1, SSPE case 1; 2, SSPE case 2; 3, AD

Electron cryo‑microscopy: image processing

Image processing was performed using RELION-4.0 [19,20], unless indicated otherwise. Raw movie frames were
gain-corrected, aligned, dose-weighted and summed intoa single micrograph. Contrast transfer function (CTF)
parameters were estimated using CTFFIND-4.1 [21].
Filaments were picked manually and segments extracted
initially with a box size of 1024 pixels. 2D classification
was used to remove suboptimal segments and separate
Type I from Type II filaments. Selected class averages forType I and Type II filaments were then re-extracted witha box size of 400 (for SSPE case 1) or 300 (for SSPE case2) pixels. Initial models were generated de novo from 2Dclass averages using relion_helix_inimodel2d [22]. Helicaltwist and rise were optimised during 3D auto-refinement.
Bayesian polishing and CTF refinement [23] were used to
improve the resolution of reconstructions of Type I filaments. Final maps were sharpened using standard postprocessing procedures in RELION and their resolutions
were calculated based on the Fourier shell correlation(FSC) between two independently refined half-maps at
0.143 [24]. Helical symmetry was imposed on the postprocessed maps using the relion_helix_toolbox program[25].
Model building and refinement

Atomic models of published CTE filament structures [26]
(PDB:6NWP; PDB:6NWQ) were docked manually in the

Results
For cryo-EM, we extracted tau filaments from the frontal
cortex of two cases of SSPE. Both individuals had measles
as children, with the clinical picture of SSPE manifesting
itself following a number of symptom-free years.
In SSPE case 1, immunohistochemistry with anti-tau
antibodies showed abundant neurofibrillary tangles thatwere stained by anti-tau antibodies specific for 3R tau,
4R tau and phospho-tau (AT8) (Fig. 1). Neurons and glial
cells with intranuclear inclusion bodies were observed
using an antibody against measles virus. Microglial cells
were activated, as shown by Iba-1 staining; the same wastrue of cytotoxic and T helper lymphocytes, as evidenced
by CD3 staining (Fig. 1). Similar abnormalities have been
described in SSPE case 2 [9].
Filaments from the sarkosyl-insoluble fractions were
decorated by anti-tau antibodies and gave the same
bands on Western blots as those from AD brains (Fig. 2).
It has previously been shown that the bands of sarkosylinsoluble tau from AD are identical to those from CTE[34]. The observed bands indicate that the filaments of
SSPE cases 1 and 2 are made of all six tau isoforms in a
hyperphosphorylated state, consistent with previous
results [9].
By cryo-EM, we show that the CTE fold of assembled
tau is characteristic of SSPE cases 1 and 2 (Fig. 3; Additional file 1: Figure S1). As in the CTE fold, two types oftau filaments were present, each with an unknown, internal density in the β-helix of the structured core. Type I
and Type II filaments are both made of two identical
protofilaments, with different interprotofilament packing, i.e. type I and type II CTE filaments are ultrastructural polymorphs. Type I comprised more than 90% of
the observed filaments and Type II less than 10%, similar
to what has been observed in CTE [26].
Discussion
SSPE and CTE share a long interval between the primary
insult (measles virus infection or repetitive head impacts
and blast waves) and the appearance of clinical symptoms. Like AD and CTE, tangle-bearing cases of SSPE arecharacterised by the presence of abundant filamentous
inclusions made of all six brain tau isoforms. Unlike AD,
CTE and tangle-bearing cases of SSPE share the formation of abundant tau inclusions in cortical layers II andQi et al. Acta Neuropathologica Communications(2023) 11:74

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Fig. 3 Cryo-EM cross-sections and structures of tau filaments from SSPE. a Cross-sections through the cryo-EM reconstructions, perpendicular
to the helical thickness and with a projected thickness of approximately one rung, are shown for SSPE cases 1 and 2. A majority of Type I and aminority of Type II tau filaments are each made of two copies of a single protofilament arranged in different ways (ultrastructural polymorphs).
Resolutions and filament percentages: SSPE case 1: Type I CTE filament 2.3 Å, 92%; Type II CTE filament 5.1 Å, 8%. SSPE case 2: Type I CTE filament
3.0 Å, 91%; Type II CTE filament 3.5 Å, 9%. Scale bar, 5 nm. b Cryo-EM density maps (grey transparent) of SSPE Type I and Type II tau filaments andthe atomic models coloured blue (Type I) and orange (Type II). c SSPE Type I (blue) and Type II (orange) filaments overlaid with CTE Type I (magenta)
and CTE Type II (magenta) filaments. The filament core extends from tau residues K274/S305-R379

III [9, 11]. We previously showed that the CTE fold differs from the Alzheimer tau fold by adopting a more
open conformation of the β-helix region, which contains
an internal density of unknown identity [26]. In the presence of NaCl, recombinant tau comprising amino acids
297–391 assembles into filaments with the CTE fold, but
in its absence, the Alzheimer tau fold forms [35].
We now find that the tau fold of SSPE is identical tothat of CTE. As in CTE, two types of filaments, eachmade of two identical protofilaments, were present in
SSPE cases 1 and 2. Western blots of sarkosyl-insoluble
tau fractions indicate that these filaments are made ofall six (3R + 4R) brain tau isoforms [12, 13, 26, 36]. For
3R + 4R tauopathies, one protofilament fold (the Alzheimer fold) is found in PART, AD, GSS, FBD and FDD, andthe second protofilament fold (the CTE fold) is found in
CTE. The present findings show that SSPE is a second
example of a 3R + 4R tauopathy with the CTE fold. It
remains to be seen if other conditions with tau inclusions
in cortical layers II and III also share the CTE fold.
Inflammation may be what CTE and SSPE have incommon. In SSPE case 2, extensive inflammatorychanges have been described, with perivascular lymphocyte infiltration, aggregates of hypertrophic astrocytes and activated microglia [9]. Here we show that thefrontal cortex from SSPE case 1 also exhibited microglial activation and lymphocyte infiltration. Immunoreactivity for measles virus was present in both casesof SSPE, even though they had undergone antiviraltreatment. In untreated cases of long disease duration,

Qi et al. Acta Neuropathologica Communications(2023) 11:74

measles virus was detected in cases with neurofibrillarylesions [37]. In treated cases, the detection frequency ofmeasles virus was decreased, even though neurofibrillary pathology was unaffected, suggesting that antiviraltherapies may not be able to suppress the progressionof tauopathy following SSPE [9]. It remains to be seen
if tau inclusions influence the clinical picture of SSPE.
Inflammatory changes also occur in CTE, where microglial cell activation is believed to increase tau pathology
and the presence of abundant CD68-positive microglial
cells has been demonstrated [38]. Moreover, translocator protein (TSPO) positron emission tomography
ligands for activated microglia have shown increased
signal in retired American football players who are at
risk for CTE [39].
It is unclear how inflammation and microglial cell
activation affect tau assembly. Microglial cell activation has been reported to promote tau assembly inmice [40–42] and it also characterizes neurodegenerative diseases with filamentous tau pathology other than
SSPE and CTE, the most studied of which is AD [43,44]. More work is required to identify the links between
neuroinflammation and tau assembly.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s40478-​023-​01565-2.
Additional file 1. Supplementary information: Table S1 and Figure S1.
Acknowledgements
This work was supported by the Electron Microscopy Facility of the MRC
Laboratory of Molecular Biology. We thank Jake Grimmett, Toby Darling and
Ivan Clayson for help with high-performance computing. We also thank Ms R.
Otani for technical assistance and Professor B. Ghetti for helpful discussions.
For the purpose of open access, the MRC Laboratory of Molecular Biology has
applied a CC BY public copyright licence to any Author Accepted Manuscript
version arising.
Author contributions
MH, MT, MS, AA, YI, HM and MY identified the patients and performed
neuropathology; MH extracted filaments and performed immunoelectron
microscopy and Western blotting; CQ performed cryo-EM data acquisitionand structure determination; SHWS and MG supervised the project and all
authors contributed to the writing of the manuscript.
Funding
This work was funded by the Medical Research Council, as part of UK Researchand Innovation (MC-UP-A025-1013 to S.H.W.S. and MC-U105184291 to M.G.).
It was also supported by the Japan Agency for Science and Technology(Crest, JPMJCR18H3, to M.H.) and the Japan Agency for Medical Research andDevelopment (AMED, JP20dm0207072, to M.H., and AMED, JP21wm0425019,
to M.T.). M.T. was supported by intramural funds from the National Center of
Neurology and Psychiatry.
Availablity of data and materials
Cryo-EM maps have been deposited in the Electron Microscopy Data Bank(EMDB) with the accession numbers EMD-16532, EMD-16535. Correspondingrefined atomic models have been deposited in the Protein Data Bank (PDB)
under accession numbers 8CAQ, 8CAX. Please address requests for materials
to the corresponding authors.

Page 6 of 7

Declarations
Competing Interests
The authors declare that they have no competing interests.
Received: 2 February 2023 Accepted: 10 April 2023

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