Induction of a transmissible tau pathology by traumatic brain injury

15 Pages·2017·1.25 MB·English

doi:10.1093/brain/awy193 BRAIN2018:Page1of15 | 1
Induction of a transmissible tau pathology by
traumatic brain injury
Elisa R. Zanier,1 Ilaria Bertani,1 Eliana Sammali,1,2 Francesca Pischiutta,1
Maria Antonietta Chiaravalloti,1 Gloria Vegliante,1 Antonio Masone,1 Alessandro Corbelli,3
Douglas H. Smith,4 David K. Menon,5 Nino Stocchetti,6,7 Fabio Fiordaliso,3 Maria-Grazia De
Simoni,1 William Stewart8,9 and Roberto Chiesa1
Traumatic brain injury is a risk factor for subsequent neurodegenerative disease, including chronic traumatic encephalop-
athy, a tauopathy mostly associated with repetitive concussion and blast, but not well recognized as a consequence of severe
traumatic brain injury. Here we show that a single severe brain trauma is associated with the emergence of widespread
hyperphosphorylatedtaupathologyinaproportionofhumanssurvivinglateafterinjury.Inparallelexperimentalstudies,in
a model of severe traumatic brain injury in wild-type mice, we found progressive and widespread tau pathology, replicating
the findings in humans. Brain homogenates from these mice, when inoculated into the hippocampus and overlying cerebral
cortex of na¨ıve mice, induced widespread tau pathology, synaptic loss, and persistent memory deficits. These data provide
evidence that experimental brain trauma induces a self-propagating tau pathology, which can be transmitted between mice,
and call for future studies aimed at investigating the potential transmissibility of trauma associated tau pathology in
humans.
1 Department ofNeuroscience, Istituto diRicerche FarmacologicheMarioNegri IRCCS, 20156Milan, Italy
2 Department ofCerebrovascular Diseases,Fondazione IRCCS – Istituto Neurologico Carlo Besta,Milan, Italy
3 Department ofCardiovascular Research, Istituto di Ricerche FarmacologicheMarioNegri IRCCS, 20156Milan, Italy
4 Penn Centre forBrainInjuryand Repair andDepartment ofNeurosurgery, Perelman School ofMedicine,University of
Pennsylvania, Philadelphia, PA19104,USA
5 Division ofAnaesthesia, Department ofMedicine, UniversityofCambridge, Cambridge CB22QQ, UK
6 Department ofPathophysiology andTransplants, University ofMilan, 20122Milan, Italy
7 Fondazione IRCCS Ca` GrandaOspedale Maggiore Policlinico di Milano, 20122Milan, Italy
8 Institute ofNeuroscience andPsychology, University ofGlasgow, Glasgow G128QQ, UK
9 Department ofNeuropathology, Queen Elizabeth UniversityHospital, Glasgow G514TF, UK
Correspondence to: Elisa R.Zanier
Department ofNeuroscience, Istituto di Ricerche Farmacologiche MarioNegri IRCCS,Via G.LaMasa19,20156 Milan, Italy
E-mail: [email protected]
Correspondence mayalso beaddressed to: Roberto Chiesa
E-mail: [email protected]
Keywords: traumatic braininjury; taupathology; prion;neurological dysfunction; neurodegeneration
Abbreviations: CTE = chronic traumatic encephalopathy; P-tau = hyperphosphorylated tau; TBI = traumatic braininjury
ReceivedJanuary15,2018.RevisedMay28,2018.AcceptedJune6,2018.
(cid:2)TheAuthor(s)(2018).PublishedbyOxfordUniversityPressonbehalfoftheGuarantorsofBrain.Allrightsreserved.
Forpermissions,pleaseemail:[email protected]
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2 | BRAIN 2018: Page 2 of 15 E. R. Zanier et al.
Introduction inducedtaupathologycouldbetransmittedbetweenmiceand
cause neurological deficits, satisfying the requirements for a
Traumatic brain injury (TBI) is a recognized risk factor for bona fide tau prion.
delayed neurodegeneration and dementia, particularly chronic
traumatic encephalopathy (CTE). The neuropathological
picture that is emerging with late survival from TBI is of a Materials and methods
complex of pathologies featuring abnormalities in tau, amyl-
oid-beta, neuronal loss, axonal degeneration, neuroinflamma-
Study design
tionandblood–brainbarrierdisruption(Johnsonetal.,2012,
2013; Hay et al., 2016). Of particular note is the emergence
The aim of the study was to test whether a single severe TBI
of hyperphosphorylated forms of the microtubule-associated was able to induce a self-propagating tau pathology. First, we
protein tau in a stereotypical pattern and distribution re- analysed the deposition of P-tau late after single TBI in
garded as pathognomonic of CTE (McKee et al., 2016). humans. Next, we characterized the temporal evolution of
Specifically, in its early stage, CTE is recognized through the tau pathology in wild-type mice subjected to severe controlled
demonstration of clusters of perivascular neurons and glia corticalimpact.Finally,wetestedwhetherauthentictauprions
containing hyperphosphorylated tau (P-tau), typically with are generated after trauma by attempting the transmission of
tauopathy to naı¨ve mice by intracerebral inoculation of brain
patchy distribution and concentrated towards the depths of
homogenates from TBI mice.
cortical sulci (Geddes et al., 1999; McKee et al., 2014, 2016;
Male C57BL/6J mice (8 to 9 weeks of age) from Envigo
Mez et al., 2017). There is a suggestion of hierarchical pro-
were used in this study. The animals were housed four per
gression of pathology from these initially focal and patchy cage at controlled temperature (22(cid:2)2(cid:3)C) and relative humid-
cortical P-tau deposits to more widespread cortical, medial
ity (60(cid:2)5%) with a 12/12-h light/dark cycle and free access
temporal and subcortical grey matter in late-stage disease to pelleted food and water.
(McKee et al., 2014). However, the mechanisms by which Allanimalexperimentsweredesignedinaccordancewiththe
this initially localized cortical pathology progresses to more ARRIVE guidelines (Kilkenny et al., 2010), with a commit-
widespread and generalized disease are still not known. ment to refinement, reduction, and replacement, minimizing
In Alzheimer’s disease, tau pathology appears to progress the numbers of mice, while using biostatistics to optimize
along neural networks, suggesting transcellular spread of tau mouse numbers [as in our previously published work using
the mouse TBI model (Zanier et al., 2016) and prion inocula-
through neural connections (Goedert et al., 2017). In vivo
tion in mice (Bouybayoune et al., 2015)]. Thus, for statistical
studies have described the spreading of Alzheimer’s disease-
validity, we used 8 to 12 mice for the behavioural tests, and
related tau pathology from local sites to distant regions of
three toeightmice forbiochemical analysis andhistology. The
the brain (Clavaguera et al., 2009, 2013; de Calignon et al.,
mice were assigned to the different experimental groups using
2012; Ahmed et al., 2014; Guo et al., 2016), and it has been
randomization lists (www.randomizer.org). Inoculations, be-
suggested that tauaggregates—or seeds—serve as the agent of havioural and histological assessments were done by re-
this spread, transmitting the aggregated/hyperphosphorylated searchers blinded to the experimental groups.
state from cell to cell in a prion-like fashion (Prusiner, 2012;
Clavaguera et al., 2013; Iba et al., 2013, 2015; Woerman
et al., 2016). Prions are proteinaceous infectious agents con- Ethics statement
sisting of misfolded prion protein (PrPSc) that propagate by
Human tissue was obtained from the Glasgow TBI Archive of
imposing their abnormal structures on host-encoded cellular
theDepartmentofNeuropathology,QueenElizabethUniversity
prionprotein(PrPC)(Prusiner,1982).AsPrPScprionscolonize
Hospital,Glasgow,UK.Tissuesampleswereacquiredatroutine
the affected patient, they perturb the function of the nervous
diagnostic autopsy, with approval from the Greater Glasgow
system, leading to motor and neurophysiological deficits, cog- and Clyde Biorepository Governance Committee for their use
nitive decline, and ultimately death (Chiesa, 2015). Whether in research. Procedures involving animals and their care were
TBI generates misfolded or aggregated forms of tau protein conducted in conformity with the institutional guidelines at the
that propagate by inducing misfolding of native tau in injured IRCCS–MarioNegriInstituteforPharmacologicalResearchin
tissue and can be transmitted from one individual to another compliance with national (D.lgs 26/2014; Authorization n. 19/
(tauprions),isnotclear(Prusiner,2012;Mudheretal.,2017). 2008-AissuedMarch6,2008byMinistryofHealth)andinter-
national laws and policies (EEC Council Directive 2010/63/UE;
We hypothesized that late P-tau deposition after severe TBI
the NIH Guide for the Care and Use of Laboratory Animals,
wouldshowprionbehaviour,andaddressedthishypothesisin
2011 edition). They were reviewed and approved by the Mario
sequential steps. First, we set out to demonstrate and charac-
Negri Institute Animal Care and Use Committee that includes
terize the deposition of P-tau late after severe TBI as distinct
ad hoc members for ethical issues, and by the Italian Ministry
from P-tau deposition in ‘normal’ aged human controls.
ofHealth(Decretono.D/07/2013-Band301/2017-PR).Animal
Second, we sought the emergence of self-propagating P-tau
facilities meet international standards and are regularly checked
pathology inwild-type mice subjectedtosevere TBI that repli- by a certified veterinarian who is responsible for health moni-
cated P-tau pathology observed in humans long after a single toring, animal welfare supervision, experimental protocols and
severe TBI. Finally, we went on to establish whether this TBI- review of procedures.
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Brain trauma induces tau prion BRAIN 2018: Page 3 of 15 | 3
Human studies tangles and neurites of sparse to moderate density in the trans-
entorhinal cortex with or without small numbers in the CA1
At the time of the original diagnostic autopsy, whole brains sector of the hippocampus; score 2, PHF1-immunoreactive
were immersion-fixed in 10% formal saline for a minimum of neurofibrillary tangles and neurites as described for score 1 to-
3 weeks, then the specimens were examined, sampled and pro- gether with neurofibrillary tangles extending into the fusiform
cessed to paraffin tissue blocks. For this study, 15 patients sur- gyrus (lateral to entorhinal cortex) and sparse tangles in the
viving a year or more from single moderate or severe, closed cortex (cingulate/insular blocks); and score 3, contained
TBI (defined as Glasgow Coma Scale at presentation 513) and PHF1-immunoreactive neurofibrillary tangles and neurites in
15 age-matched controls with no history of TBI or of known more widespread sectors of the hippocampus and subiculum,
neurological disease were randomly selected from the compre- with extensive medial temporal, cingulate and insular cortical
hensive databases of the Glasgow TBI Archive by a laboratory involvement.
researchassistantwithnoknowledgeofstudypurposeordesign
or of the pathologies in each of the TBI cases and controls
Mouse model of traumatic brain
selected. Full demographic information for late TBI cases and
controls is provided in Supplementary Table 1. Information on injury
TBIandcontrols’historywasobtainedfromreviewofdiagnos-
Thecontrolledcorticalimpactbraininjurymousemodelusedin
tic post-mortem and/or forensic reports. The TBI survival cases
this study replicates both the mechanical forces and the main
andtheage-matchedcontrolshadnorecordedhistoryofexpos-
secondary injury processes observed in severe TBI patients with
ure to contact sports or military service. The mechanism of
brain contusionandisassociated with clinically-relevant behav-
injury is summarized in Supplementary Table 1. None were
ioural and histopathological outcomes (Smith et al., 1995;
penetrating. From review of the original hospital records, at
Pischiutta et al., 2018). Mice were anaesthetized by isoflurane
presentation, intracerebral haemorrhage was present in five
inhalation (induction 3%; maintenance 1.5%) in an N O/O
cases, subdural a further seven, with three cases having no evi- 2 2
(70%/30%) mixture and placed in a stereotaxic frame. Rectal
dence of intracranial haemorrhage. Following the original TBI,
temperature was maintainedat 37(cid:3)C.Mice were then subjected
all cases were discharged from hospitalization following recov-
to craniectomy followed by induction of controlled cortical
ery and,ultimately,diedfrom causesofdeathunrelatedtoTBI.
impact brain injury as previously described (Zanier et al.,
None were in a persistent vegetative state prior to death. Each
2016). Briefly, the injury was induced using a 3-mm rigid im-
case was sampled and assessed for this study as standardized
pactor driven by a pneumatic piston rigidly mounted at an
regions, no matter the injury severity or location. As this study
angleof20(cid:3) fromtheverticalplaneandappliedto the exposed
accesses archival tissue blocks, there were necessary restrictions
dura mater, between bregma and lambda, over the left parieto-
on availability of sampled regions. Nevertheless, the standar-
temporal cortex (antero-posteriority: (cid:4)2.5mm, laterality:
dizedsamplingprotocolsusedattheoriginaldiagnosticautopsy
(cid:4)2.5mm), at an impactor velocity of 5m/s and deformation
includedacoronalsliceofthecerebralhemispheresatmid-thal-
depth 1mm, resulting in a severe level of injury (Smith et al.,
amiclevel from whichtissueblocks frommultiplebrainregions
1995). The craniotomy was then covered with a cranioplasty
were selected for the purposes of this study to include: the
and the scalp sutured. Sham mice received identical anaesthesia
corpus callosum with adjacent cingulate gyrus; the medial tem-
and surgery without brain injury.
poral lobe to include the hippocampus and entorhinal cortex;
the insular cortex and the thalamus. From these tissue blocks
8mm sections were prepared for immunohistochemistry proced- Transmission studies
ures. After deparaffinization and rehydration, endogenous per-
oxidase was quenched by immersing sections in 3% aqueous Brainhomogenates(10%w/vinPBS)fromtheipsi-andcontra-
H O for15min.Thereafter,heat-inducedantigenretrievalwas lateralcerebralcortex(includingallthecorticaltissueabovethe
2 2
performed using a microwave pressure cooker for 8min in pre- rhinalfissure)ofmice12monthsfromTBIorshaminjurywere
heated0.1MTrisEDTAbuffer(pH8),followedbyblockingin inoculated in the hippocampus (A/P, (cid:4)2.5mm from bregma; L,
50ml of normal horse serum (Vector Labs) per 5ml of (cid:2)2.0mm; D/V, (cid:4)1.8mm) and overlying cerebral cortex (A/P,
OptiMaxTM buffer (BioGenex) for 30min. The sections were (cid:4)2.5mm from bregma; L, (cid:2)2.0mm; D/V, (cid:4)0.8mm). Inocula
then incubated in antibody to phosphorylated tau (PHF1; were from single brains. The TBI brain homogenates showing
1:1000; gift from P. Davies, Feinstein Institute for Medical the highest amounts of P-tau by western blot were chosen for
Research,Manhasset, NY,USA)at 4(cid:3)C for20hpriortowash- inoculation.Micewereinjectedbilaterally(2.5mleach,injection
ing and application of biotinylated secondary antibody for speed of 1.25ml/min).
30min,followedbyanavidinbiotincomplex,asperthemanu-
facturer’s instructions (Vectastain Universal Elite Kit, Vector Novel object recognition
Labs). The target antigen was visualized using the DAB perox-
idase substrate kit (Vector Labs), with the sections then coun- Mice were tested in an open-square grey arena (40(cid:5)40cm),
terstained with haematoxylin. Control sections (positive and 30cm high, with the floor divided into 25 squares by black
negative) for each antibody were run in parallel with the test lines. The following objects were used: a black plastic cylinder
sections to confirm antibody specificity. Sections were viewed, (4(cid:5)5cm), a glass vial with a white cap (3(cid:5)6cm) and a
blindtowhethertheywereTBIorcontrol,usingaLeicaDMRB metal cube (3(cid:5)5cm). The task started with a habituation
light microscope (Leica Microsystems) and the extent and dis- trial during which mice were placed in the empty arena for
tribution of tau pathology was assessed and semiquantitatively 5min and their movements were recorded as the number of
scoredas:score0,wherenotaupathologywaspresent;score1, line-crossings. The next day, mice were placed in the same
PHF1 pathology comprising immunoreactive neurofibrillary arena containing two identical objects (familiarization phase).
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Exploration was recorded in a 10-min trial by an investigator positive pixels/total pixels, and reported as the percentage
blinded to the experimental group. Sniffing, touching and stained area for statistical analysis.
stretching the head toward the object at a distance of no For immunofluorescence, brain sections on glass slides were
more than 2cm were scored as object investigation. Twenty- boiledincitratebufferandincubatedinblockingsolutionwith
four hours later (test phase) mice were placed in the arena the following primary antibodies: anti-drebrin (Fitzgerald,
containing two objects: one identical to one of the objects 1:200) an actin-binding protein enriched in dendritic spines,
presented during the familiarization phase (familiar object), (Sekino et al., 2017) and anti-vesicular glutamate trans-
and a new, different one (novel object), and the time spent porter-1 (VGLUT-1, Synaptic System, 1:6000), for analysis
exploring the two objects was recorded for 10min. Memory of the presynaptic compartment (Senatore et al., 2012). Slices
was expressed as a discrimination index, i.e. the time spent were then incubated with biotinylated secondary antibody
exploring the novel object minus the time spent exploring (Vecstain, 1:500) and streptavidin 647 (Molecular Probes,
the familiar one, divided by the total time spent exploring 1:500), and examined by confocal microscopy (FV-500
both objects; the higher the discrimination index, the better Olympus) acquiring images in two different areas of CA1
the performance. field of the hippocampus using a 60(cid:5) oil objective. Z-stack
images were acquired: 0.2mm Z-distance, 3-mm thick confocal
z-stacks (optical section depth 0.2mm, 21 sections/z-stack) and
Immunohistochemistry and 1024(cid:5)1024 pixel resolution. Confocal images were deconvo-
immunofluorescence luted (CellP) and puncta were identified with Imaris software
using the ‘quality’ parameter (Heise et al., 2016). Only puncta
Mice were euthanized by cervical dislocation, brains were stringently conforming with the quality parameter entered
removed and fixed in 10% formalin for 24h, dehydrated in analysis.
graded ethanol solutions, cleared in xylene, and embedded in
paraffin. Serial sections (5mm thick) were cut onto glass slides
and boiled in citrate buffer (10mM pH 6.0, Dako) for antigen Biochemical analysis
retrieval, followed by incubation with the following anti-tau
monoclonal antibodies: AT8 (phospho-tau Ser202/Thr205; Frozen brain tissue was homogenized in 10 volumes of phos-
Thermo Fisher; 1:1000), AT180 (phospho-tau Thr231; Thermo phate-bufferedsaline(PBS).Homogenateswerealiquotedand
Fisher; 1:1000), PHF1 (Ser396/Ser404; from P. Davies; 1:4000) stored at (cid:4)80(cid:3)C until use. Western blot analysis of total
andMC1(aa312–322;fromP.Davies;1:100).AT8andAT180 brain homogenates with monoclonal antibody AT8 was
immunostaining was visualized using the ARK kit (Dako). For done on heat-stable protein fractions prepared according to
secondarystainingofMC1andPHF1,sliceswereincubatedwith Petry et al. (2014) to avoid the interfering signal due to en-
biotinylated goat anti-mouse IgG1 in Superblock Solution dogenous immunoglobulins. Proteins (50mg) were separated
(Pierce), followed by visualization with the TSA Biotin System onNuPAGE4–12%gels(ThermoScientific)andblottedonto
(Perkin Elmer) and DAB (diaminobenzidine) Substrate nitrocellulose membranes (Hybond, GE Healthcare). After
Chromogen System (Dako). blocking with 5% dry milk for 1h at room temperature,
Slices were examined by an Olympus BX-61 Virtual Stage membranes were incubated overnight at 4(cid:3)C with anti-total
microscope, interfaced with VS-ASW-FL software (Olympus). tau(1:10000,Dako) orAT8(1:500,ThermoScientific),then
For quantification of the AT8-positive staining in TBI mice with HRP-conjugated secondary antibodies (Santa Cruz).
regions of interest were selected according to the Paxinos Peroxidase activity was detected using an ECL (Iluminata
atlas (Paxinos and Franklin, 2004) in coronal (bregma Forte) kit (Millipore) and visualized with a ChemiDoc ima-
(cid:4)1.6mm) and sagittal (lateral 1.5mm) sections. In TBI mice ging system (Bio-Rad). For RIPA insolublepreparation 100ml
the ipsilateral cortex was analysed over an area 1mm deep of brain homogenate were incubated for 20min on an orbital
from the edge of the contusion (Pischiutta et al., 2018) and shaker with 1% RIPA buffer (50mM Tris-HCl, pH 7.4, 1%
the contralateral from midline to the entorhinal cortex. In the NP-40, 150mM NaCl, 0.25% Na-deoxycholate, 1mM
inoculated mice, regions of interest included whole cortex, EDTA, 1mM Na VO , 1mM NaF, 1mM PMSF and prote-
3 4
hippocampus, thalamus and cerebellum. The same regions of ase inhibitors). Brain extract was ultracentrifuged for 60min
interest were selected in sham mice. Images were acquired at100000gatroomtemperature. Thepelletwasresuspended
using a 60(cid:5) oil objective with pixel size 0.115mm. in 450ml of RIPA buffer and ultracentrifuged at 100000g for
Acquisition was done over 5-mm thick stacks, with step size 30min at room temperature. The supernatant was carefully
1mm. The different focal planes were merged into a single removed and the pellet was resuspended in 100ml of 1(cid:5)
stack by mean intensity projection to ensure consistent focus Laemmli sample buffer and proteins analysed by western
throughout the sample. Images were analysed using Fiji soft- blot.
ware (http://fiji.sc/Fiji). For assay of protease resistance brain lysates prepared in
The digital separation of DAB staining from haematoxylin 0.05% TritonTM X-100 in PBS were centrifuged at 300g for
signal was done by colour deconvolution (http://www. 5min. Proteins (20mg) in the supernatants were digested
mecourse.com/landinig/software/cdeconv/cdeconv.html) and with pronase (0–10mg/ml; Sigma) in a final volume of
the DAB channel was used for quantification. To ensure con- 20ml for 1h at 37(cid:3)C, then resolved by NuPAGE 4–12%
sistency of the segmentation in the image processing, we sub- gels and analysed by western blot with primary anti-tau
tracted the intensity background from the image calculated by antibody BR135 (1:1000, gift from M. Goedert, MRC
averaging non-specific signal in identified region of interest. Laboratory of Molecular Biology, Cambridge, UK; Goedert
Specific signal after the image processing was computed as et al., 1989).
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Brain trauma induces tau prion BRAIN 2018: Page 5 of 15 | 5
Electron microscopy more,PHF1-immunreactiveprofilesappearedasneurofibrillary
tangles and immunoreactive neurites, astrocytes and plaques
Three controls and three TBI inoculated mice were deeply within all cortical layers, although often with higher density
anaesthetized and perfused through the ascending aorta with
in the superficial cortical layers (Fig. 1A). Elsewhere, where
PBS (0.1M; pH 7.4) followed by a 5-min perfusion with 4%
hippocampal profiles were present, all sectors appeared
paraformaldehyde.Hippocampuswasexcisedandcutinsagittal
involved, CA2 less frequent than other sectors in this series.
planewitharazorbladeandpostfixedin4%paraformaldehyde
Sixcasesshowedinvolvementofstructuresintheregionofthe
and2%glutaraldehydeinphosphatebuffer0.12M,thenfor2h
thalamusassparseimmunoreactivethalamicornigralneurons,
in OsO . After dehydration in graded series of ethanol, tissue
4
samples were cleared in propylene oxide, embedded in epoxy occasional neurites and immunoreactive astrocytes. Although
medium (Epoxy Embedding Medium kit; Sigma-Aldrich) and the proportions of patients with PHF1-immunoractive tau
polymerized at 60(cid:3)C for 72h. From each sample, one semi- pathology were similar in controls and TBI (12 cases with
thin (1mm) section was cut with a Leica EM UC6 ultramicro- pathology in each group), the extent and distribution of the
tome and mounted on glass slides for light microscopic inspec- pathologieswereconsiderablygreaterinsurvivorsofTBIthan
tiontoidentifythestratumoriensofthehippocampus.Ultrathin
in non-injured controls (Fig. 1B). Specifically, in controls,
(70-nm thick) sections of the areas of interest were obtained,
PHF1-immunoreactive neurofibrillary tangles and neurites
counterstained with uranyl acetate and lead citrate, and exam-
were largely restricted to sparse profiles in the entorhinal
ined with an Energy Filter Transmission Electron Microscope
cortex, with just four cases showing more extensive patholo-
(Libra120, Carl Zeiss NTS) equipped with an yttrium alumin-
gies extending to the hippocampus and cortex. In contrast, in
ium garnet scintillator slow-scan charge-coupled device camera
(Sharpeye,TRS).Aregionofinterestincludingthepostsynaptic late survivors of TBI, 9 of 12 PHF1-immunoreactive cases
density was manually traced and its area and thickness mea- displayedhigherstagetaupathology,withPHF1-immunoreac-
sured by iTem software (Olympus Soft Imaging Solutions, tive profiles in moderate to high density across the medial
Germany) of 263 synapses in control inoculated mice and 262 temporal lobe, including the hippocampus, and extending to
inTBIinoculatedmicefittingthefollowingtwocriteria:(i)pres- the cortex. Neuritic P-tau-positive profiles were found in the
ence of a cluster of presynaptic vesicles (at least three of them); neuropil, many with a truncated, granular appearance as illu-
and (ii) presence of a defined synaptic cleft.
strated in Fig 1A. In 3 of 12 TBI cases with P-tau pathology,
immunoreactive neuronal and glial profiles showed focal clus-
Statistical analysis
tering around cortical vessels, in two of these at the depths of
sulci (Fig. 1A); appearances consistent with the pathogno-
All graphs represent the mean(cid:2)standard error of the mean
monic pathology of CTE described in the preliminary NIH
(SEM). GraphPad Prism 7 was used for statistical analyses.
diagnostic criteria (McKee et al., 2016). One further TBI
A categorical assessment score was used to assess human tau
deposition.Resultsweredichotomizedineither‘low’score(0–1) case, an 87-year-old male with a 5-year survival following a
or ‘high’ score (2–3) and the chi-square test was used to assess roadtraffic accident, showedclustersof P-tauimmunoreactive
differencesbetweengroups.Datainmicewereanalysedbyone- subpialastrocytesin the medialtemporal lobe, consistent with
way ANOVA, followed by Tukey’s test to assess differences in ageing-relatedtauastrogliopathy(Kovacsetal.,2016).Bothin
case of three or more groups (behavioural tests and temporal late TBI cases and controls the extent of P-tau pathology ap-
changesoftaudeposition)orbyunpairedt-test.P-values50.05 peared to increase with increasing age; the higher score path-
were considered to be statistically significant.
ologyencounteredmoreoftenwithincreasingage.Specifically,
the four controls with higher tau scores (2 or 3) were among
Data availability
the older aged (74 years or above) controls. Of note, while
again typically olderwithinthe cohort, the nine late TBI cases
The authors confirm that the data supporting the findings of this
with higher tau scores ranged from a younger age (age 59
studyareavailablewithinthepaperanditsSupplementarymaterial.
years upwards) than controls. No clear association between
survival duration and extent of tau pathology could be ascer-
Results tained in this small series (Supplementary Fig. 1). In keeping
with injury severity, evidence of healed contusion was near
ubiquitous; present in all but one case. There was no associ-
Phosphorylated tau deposition is
ationbetweenpresenceornature ofintracranialhaemorrhage,
widespread in patients surviving a
including contusions, and tau pathology.
year or more from single TBI
Single severe TBI induces a
Representative tissue sections from the brains of patients sur-
progressive tau pathology that
viving a median of 5 years (range: 1–18 years) following a
single moderate or severe, closed TBI [n=15; age 19–89 spreads from the site of injury
(median 60) years], or control subjects with no history of
in wild-type mice
TBI or known neurological disease [n=15; age 20–92
(median 60) years] (Supplementary Table 1) were stained for We tested whether single focal brain trauma triggered propa-
P-tauusingantibodyPHF1.InTBIpatientssurvivingayearor gating tau pathology in wild-type mice. Two-month-old
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6 | BRAIN 2018: Page 6 of 15 E. R. Zanier et al.
Figure1 TaupathologyinsurvivorsofasinglemoderateorsevereTBI.(A)Typically,controlsshowedsparseP-tauimmunoreactive
neurofibrillarytanglesandneuriteslocalizedtotheentorhinalcortex(i;59-year-oldmale,nohistoryofTBI).Incontrast,patientssurvivingayear
ormorefromsinglemoderateorsevereTBIshowedmoreextensiveneurofibrillary,neuriticandglialP-tauimmunoreactiveprofiles,with
occasionalclustersaroundcorticalvesselsina‘CTE-like’manner(ii;59-year-oldmale17-yearsurvivalfromsinglesevereTBI),andextending
beyondtheentorhinalcortextoinvolvewidergreymatterregionsincludingthehippocampus(iii;87-year-oldmale5-yearsurvivalfromsingle
severeTBI).(B)WhiletheproportionofpatientsinTBIandcontrolswithtaupathologieswassimilar,followingTBItheextentanddistributionof
pathologywereconsiderablygreater.Thus,usingasemiquantitativeschemetoscoretaudistribution,whileallbutfourcontrolsshowednoor
onlylocalizedtaupathology(score0or1),inamajorityofTBIcases(9of15)P-taupathologywaswidespread(score2or3;P=0.0035;(cid:2)2).All
sectionsstainedforPHF-1.Scalebar=100mm.
C57BL/6J mice were subjected to severe TBI (16 mice) or to to injury, withconcentration tothe regions adjacent to injury
sham injury (14 mice). Eight TBI and seven sham mice were [Fig. 2A (cf. i and ii), B, and Supplementary Fig. 2]. In con-
sacrificed at 3 months and used for immunohistochemistry trast, at 12 months, bothipsi- andcontralateral P-tau pathol-
(four TBI and four sham) or biochemical analysis (four TBI ogies were present, supporting progressive spreading of tau
and three sham). One TBI mouse was found dead at pathologyfromtheinjuredhemisphere[Fig.2A–D(cf.Aiand
(cid:6)4 months, and the brain was not collected due to prolonged Aiii;cf.CivandCvi)andSupplementaryFig.2].Westernblot
post-mortem delay. The remaining mice were culled at analysis found increased levels of total and AT8-reactive tau
12 months and used for immunohistochemistry (four TBI inonefrozenTBIbraincomparedtosham-injuredcontrolsat
andfivesham)andforbiochemistry(threeTBIandtwosham). 3 months and in two of three TBI brains at 12 months (an
Phospho-tau immunopositivity, assessed by AT8, AT180 example is shown in Supplementary Fig. 3). Thus, three of
and PHF1 antibodies, was evident in two of four mice at seven TBI mice ((cid:6)40%) developed pathological tau at
3monthspost-TBIandrestrictedtothehemisphereipsilateral 3 months, and six of seven ((cid:6)85%) at 12 months.
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Brain trauma induces tau prion BRAIN 2018: Page 7 of 15 | 7
Figure2 EvidenceoftaupathologyinmouseTBI.AT8immunostainingandquantificationoftheAT8-positiveareaintheipsilateraland
contralateralcortexofshamandTBImiceat3and12monthspost-injury.(AandB)AT8immunoreactivityisdetectedintheipsilateral(ipsi)
pericontusionalcorticalareaintwooffourmiceat3monthspost-TBI,andinallmiceat12months.(CandD)InthecontralateralcortexAT8
immunoreactivityisdetectedonlyafter12monthspost-TBI.Dataaremean(cid:2)SEM;n=4–5;*P50.05;**P50.01byone-wayANOVA,Tukey
posthoctest.Scalebar=100mm(BandD).
P-tau was observed as neuronal cytoplasmic, neuritic and prion, causing brain dysfunction, we intracerebrally inocu-
glial profiles throughout the cerebral cortex, hippocampus lated 10% brain homogenates from TBI and sham-injured
(CA1andCA3 fields)andthalamus,with pathologyalso in mice into na¨ıve C57BL/6J mice. Brain homogenates of con-
the striatum and zona incerta (Fig. 3). In addition, a not- tused and contralateral hemispheres (TBI and TBI ,
ipsi contra
able observation was numerous small to medium sized, respectively) showing increased levels of total and AT8-posi-
rounded, ‘grain-like’ profiles, particularly within the neuro- tive tau by western blot at 12 months post-TBI
pil (Fig. 3C). Occasionally there was faint clustering of (Supplementary Fig. 3) were inoculated bilaterally into the
these granular immunoreactive profiles (Fig. 3G), with hippocampus and overlaying cerebral cortex of the recipient
still other examples showing apparent localization along mice (Fig. 4A). The mice were examined in the novel object
cortical vessels (Fig. 3H). Elsewhere, glial cytoplasmic P- recognition test 8 and 12 months post-inoculation (m.p.i.).
tau immunoreactivity was present within white matter of There was an overt memory deficit in the TBI - but not in
ipsi
the corpus callosum. the TBI -inoculated mice at 8m.p.i. (Fig. 4B). At 12
contra
m.p.i the memory deficit persisted in the TBI - and was
ipsi
also detected in the TBI -inoculated mice (Fig. 4C). No
Tau prions are generated in TBI contra
memory deficit was seen in mice inoculated with sham-
mice, causing transmission of injured brain tissue.
tau pathology, synaptic loss and This was confirmed in an independent experiment in
which we tested the effect of the inoculation of TBI brain
behavioural deficits
homogenate, obtained from the injured hemisphere
The evidence that TBI induced a P-tau pathology that pro- 12 months post-injury, on recognition memory in the
gressively spread throughout the brain suggested forma- inoculated animals at an earlier time (4m.p.i). Memory
tion of a self-templating tau conformation. To test was already significantly impaired at 4 months, and this
whether this could be horizontally transmitted like a was confirmed at 8m.p.i. (Fig. 5B and C). Locomotor
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8 | BRAIN 2018: Page 8 of 15 E. R. Zanier et al.
Figure3 Widespreadtaupathologyinmice12monthspost-TBI.ExamplesoftauimmunostainingwiththeAT180(AandF),PHF1(B),
andAT8(C–E,GandH)antibodiesinthecerebralcortex(AandB),CA1(C)andCA3(D)fieldsofthehippocampus,andinthezonaincerta
(EandF).Examplesofclustersofsmalltomediumsized,rounded,‘grain-like’profiles,inthezonaincerta(G),andgranularprofilesalonga
corticalvessel(H;arrows).Scalebars:20mm(A–DandG);10mm(EandF);50mm(H).
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Brain trauma induces tau prion BRAIN 2018: Page 9 of 15 | 9
Figure4 Inoculationofcontusedtissuesinducesmemorydeficitsinwild-typemice.(A)Groupsofmiceusedintheinoculation
studies.C57BL/6Jmalemicewereeitherleftuntreated(openbar)orinoculatedbilaterallyintothehippocampusandoverlayingcerebralcortex
with10%brainhomogenatesfromshammice(grey)orfromthecontused(TBI ,indigo)orcontralateral(TBI ,magenta)hemisphereat
ipsi contra
12monthspost-TBI.(BandC)Recognitionmemorywasinvestigatedbythenovelobjectrecognition(NOR)testat8(B)and12(C)months
afterinoculation.Performanceinthenovelobjectrecognitiontaskwasexpressedasdiscriminationindex(thehigherthediscriminationindexthe
bettertheperformance).(DandE)Locomotoractivitywassimilarinna¨ıve,shamandTBIinoculatedmiceat8(D)and12(E)monthsafter
inoculation.Dataaremean(cid:2)SEM;*P50.05,versusna¨ıve;#P50.05versusshambyone-wayANOVA,Tukeyposthoctest.
activity was similar in sham and TBI inoculated mice, indi- with impaired synaptic efficacy. Analysis of P-tau deposition
catingthatthedeficitinthenovelobjectrecognitiontestwas by antibody AT8 showed a significant difference between
not due to motor abnormalities (Figs 4D, E, 5D and E). sham and TBI inoculated mice not only at the site of injec-
The memory deficit was associated with hippocampal tion, but also widespread throughout the brain (Fig. 7).
synaptic pathology. Analysis of pre- and postsynaptic com- Specifically, P-tau neurons, neurites and glia were identi-
partments by quantitative VGLUT-1 and drebrin immuno- fied in the cerebral cortex, hippocampus (CA1, CA3, and
fluorescence showed marked synaptic loss in the stratum hilus of the dentate gyrus) and thalamus, with morpholo-
oriens of the CA1 region at 12m.p.i. (Fig. 6A and B). gies similar to those observed in mice exposed to TBI,
Electron microscopy examination showed a significant de- including widespread ‘grain-like’ profiles (Fig. 8B–I). In the
crease in postsynaptic density thickness and area in hippo- cerebellum, P-tau immunoreactive cytoplasmic and neuritic
campal synapses in TBI inoculated mice (Fig. 6C) consistent inclusions were noted in association with Purkinje cells and
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10 | BRAIN 2018: Page 10 of 15 E. R. Zanier et al.
notable pattern and distribution. Further, a single severe
TBI induced persistent and evolving tau pathology in
wild-type mice, with characteristics similar to late post-
TBI tau pathology in humans, which progressively spread
from the site of injury to ipsi- and contralateral brain re-
gions. This TBI generated tau pathology could be trans-
mitted to wild-type recipient mice by intracerebral
inoculation, inducing tau aggregation, memory deficits
and synaptic alterations. These results indicate that tau
prions are generated in TBI, providing a mechanistic ex-
planation for how a biomechanical insult might trigger
self-sustained neurodegeneration.
In the past decade, there has been growing recognition
that repetitive mild TBI might set in motion brain patho-
logical processes that place exposed individuals at risk for
neurodegenerative disorders in mid-to-late life, particularly
CTE. While there is also evidence that tau pathology may
beseenmanyyears afterasingle severe TBI,mostattention
has focused on the association between repeated mild TBI
and CTE, with the common impression tau pathology is
uniquely associated with repetitive mild TBI, which dictates
a distinct neurodegenerative disease.
Herewe confirm that widespread CTE-like tau pathology
is present in survivors of a year or more after just a single
moderate to severe TBI in humans, and in mice exposed to
severe TBI surviving up to 1 year from injury. In our series
of 15 patients who survived a year or more post-TBI, 12
Figure5 Memorydeficitsarealreadydetectedat4
showed tau pathology, the extent and distribution of which
monthspost-inoculation.(A)Schemeofthegroupsofmice
usedintheinoculationstudies.C57BL/6Jmalemicewereeitherleft was considerably greater than that seen in age-matched,
untreated(openbar)orinoculatedbilaterallyintothehippocampus non-TBI controls. Among this small series of late post-
andoverlayingcerebralcortexwith10%brainhomogenatesfrom TBIpatientsthistaupathologyappearedtofollowastereo-
sham(grey)orTBI(contusedhemisphere,indigo)mice12months typic pattern of deposition, reminiscent of hierarchical P-
post-injury.(BandC)Recognitionmemorywasinvestigatedby tau deposition in other neurodegenerative diseases (Braak
novelobjectrecognition(NOR)testat4(B)and8(C)months and Braak, 1995; Kovacs, 2015). These data provide one
afterinoculation.Histogramsindicatethediscriminationindex(the
possible neuropathological substrate for the increased inci-
higherthediscriminationindexthebettertheperformance).(Dand
dence of neurodegenerative disease in patients who survive
E)LocomotoractivitywassimilarinshamandTBIinoculatedmice
a single moderate or severe TBI. Although the extent and
at4(D)and8(E)monthsafterinoculation.Dataaremean(cid:2)SEM,
n=11–14.***P50.001,**P50.01versusna¨ıve;##P50.01versus distribution of P-tau pathology was greater in late TBI sur-
vivors, it was not possible to establish a clear association
sham;byone-wayANOVA,Tukeyposthoctest.
between survival interval and extent of tau pathology.
Further, our data in this limited human study show similar
percentages of cases with tau pathology in TBI and control
within the granule cell layer (Fig. 8J–K). Consistent with groups, making it hard to establish whether TBI accelerates
widespread tau pathology, there was an increase in the a pre-existing age-related tau pathology, or triggers de
amount of aggregated tau in the thalamus of TBI-inoculated novo tau deposition.
mice(SupplementaryFig.4AandB).Thetauconformerthat A better understanding of the biology that underlies this
accumulated in the brains of TBI-inoculated mice was par- process calls for tractable experimental models. Previous
tially resistant to protease digestion, yielding a pronase-re- investigations have shown that TBI accelerates genetically
sistant fragment of (cid:6)10kDa (Supplementary Fig. 4C), determined tau deposition in transgenic mice overexpres-
consistent with tau prion formation (Sanders et al., 2014; sing mutant or wild-type human tau (Yoshiyama et al.,
Kaufman et al., 2016). 2005; Tran et al., 2011a, b; Ojo et al., 2016), and can
lead to tau pathology in wild-type mice (Goldstein et al.,
2012; Kondo et al., 2015; Meabon et al., 2016). A few
Discussion studies reported formation of tau aggregates shortly after
severe TBI in rats (Hawkins et al., 2013; Gerson et al.,
The present study found that a single moderate or severe 2016), with only one study showing persistent tau path-
TBI in humans resulted in widespread tau pathology with a ology in wild-type mice (Kondo et al., 2015). Our data
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