Abstract
Traumatic brain injury (TBI), characterized by acute neurological dysfunction, is one of the best known environmental risk factors for chronic traumatic encephalopathy and Alzheimer’s disease, the defining pathologic features of which include tauopathy made of phosphorylated tau protein (P-tau). However, tauopathy has not been detected in the early stages after TBI, and how TBI leads to tauopathy is unknown. Here we find robust cis P-tau pathology after TBI in humans and mice. After TBI in mice and stress in vitro, neurons acutely produce cis P-tau, which disrupts axonal microtubule networks and mitochondrial transport, spreads to other neurons, and leads to apoptosis. This process, which we term ‘cistauosis’, appears long before other tauopathy. Treating TBI mice with cis antibody blocks cistauosis, prevents tauopathy development and spread, and restores many TBI-related structural and functional sequelae. Thus, cis P-tau is a major early driver of disease after TBI and leads to tauopathy in chronic traumatic encephalopathy and Alzheimer’s disease. The cis antibody may be further developed to detect and treat TBI, and prevent progressive neurodegeneration after injury.
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Acknowledgements
We thank T. Hunter and M. Zeidel for advice; S. Hagen for Microscopy Facility (NIH grant S10 RR017927) and P. Davies for tauopathy antibodies. C.-H.C., Y.-M.L., J.A.D. and S.W. are recipients of NIA-funded T32 Translational Research in Aging Training Grant, National Science Council Postdoctoral Fellowship from Taiwan, a VA Career Development Award, and Susan G. Komen postdoctoral fellowship, respectively. R.M. is supported by Boston Children’s Hospital Pilot Grant Award and NIH training grant T32HD040128, and A.P.-L. and W.M. by NFLPA. The CTE and blast samples used are supported by grants from NIH (UO1NS086659-01, P30AG13846), VA, Sports Legacy Institute, Andlinger Foundation, NFL and WWE. The work is supported by NIH grants R01AG029385, R01CA167677, R01HL111430 and R01AG046319, and Alzheimer's Association grant DVT-14-322623 to K.P.L. and BIDMC and NFLPA pilot grants to K.P.L. and X.Z.Z.
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Contributions
A.K. and K.S. designed the studies, performed the experiments, and wrote the manuscript; R.M. helped design and conduct experiments and analysed the data on impact TBI mouse models and wrote the manuscript; J.Q. and W.M. helped with impact TBI experiments, J.M. and L.E.G. helped with blast TBI experiments and edited the manuscript; A.C.M. provided human brains and edited the manuscript; Y.S. and A.Ro. performed field excitatory postsynaptic potential (fSPSP) recording; C.-H.C., Y.Y., Y.-M.L, J.A.D., S.W., M.-L.L., O.A. and P.H. provided technical assistance; A.Ry. provided assistance for developing mAbs; A.P.-L. advised the project; X.Z.Z. originally discovered the procedures for generating cis and trans antibodies; and X.Z.Z. and K.P.L. conceived and supervised the project, designed the studies, analysed the data, and wrote the manuscript.
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Competing interests
Competing Financial Interests. K.P.L. and X.Z.Z. are inventors of Pin1 technology, which was licensed by Beth Israel Deaconess Medical Center to Pinteon Therapeutics. Both K.P.L. and X.Z.Z. own equity in, and consult for, Pinteon. K.P.L. also serves on its Board of Directors. Their interests were reviewed and are managed by Beth Israel Deaconess Medical Center in accordance with its conflict of interest policy.
Extended data figures and tables
Extended Data Figure 1 Characterization of cis and trans P-tau mAbs and robust cis P-tau in human CTE brains.
a, b, Characterization of the specificity of cis and trans P-tau mAbs by ELISA. Cis (a) and trans (b) antibodies at various concentrations were incubated with cis (pT231-Dmp), trans (pT231-Ala), cis + trans (pT231-Pro) or T231-Pro tau peptides, followed by detecting the binding by ELISA. Representative examples of ELISA are shown from 3 independent experiments. pT231-Pro, CKKVAVVRpT(Pro)PKSPSSAK; pT231-Pip, CKKVAVVRpT(homoproline)PKSPSSAK; pT231-Ala, KVAVVRpT(alanine)PKSPS; pT231-Dmp (KVAVVRpT(5,5-dimethyl-l-proline)PKSPS). c, Determination of the isotypes of cis and trans P-tau mAbs. Isotypes of cis and trans mAb heavy and light chains were determined by ELISA assay using a commercially available assay kit. d, e, Characterization of the specificity of cis and trans P-tau mAbs by immunoblotting and immunofluorescence. Brain lysates (d) or sections (e) prepared from tau-deficient (KO) or wild-type tau-overexpressing (TG) mice were subjected to immunoblotting or immunofluorescence with cis and/or trans antibody. The cis and trans signals were readily detected in TG, but not at all in KO mouse brains, with cis in the soma and neurites (pink arrow), but trans only in the soma (yellow arrow) (insets). Similar results were observed in at least three different animals. Cis, red; trans, green; DNA, blue. f–h, Robust cis P-tau in human CTE brains. 16 CTE brain tissues and 8 healthy controls were subjected to immunofluorescence, with one representative image from each case being shown) (f, g). Yellow arrow points to a neuron expressing both cis (red) and trans (green) P-tau, while pink one to a neuron expressing only trans in the soma. Fluorescence immunostaining intensity of cis P-tau was quantified using Volocity 6.3 from Perkin Elmer (h). The results are expressed as means ± s.d. and P values determined using the Student’s t-test.
Extended Data Figure 2 Colocalization of cis P-tau with other tau epitopes and its concentration near blood vessels in CTE brains.
a, b, Colocalization of cis P-tau with other tau epitopes in CTE brains. CTE brain tissues and healthy controls were stained with cis mAb and AT180, AT8, AT100, Alz50 or T22 antibodies, or trans mAb and T22 antibodies, with two examples being shown (a), and then quantified their colocalization using Coloc 2, with the results being expressed in a percentage (mean ± s.d.) (b). N.D., not detectable. c, CTE brain tissues and healthy controls were stained with cis mAb, with two examples being shown. Cis is more prominent near blood vessels, which corresponds to the typical perivascular distribution of P-tau in CTE. d, CTE brain tissues and healthy controls were stained with cis mAb (red) and the dendritic marker MAP2 (green), along with DNA dye (blue). Colours in the text correspond to their fluorescence labels. n = 4.
Extended Data Figure 3 TBI induces cis P-tau in a severity- and time-dependent manner long before other known tauopathy epitopes.
a–c, Severity- and time-dependent induction of cis P-tau after TBI. Quantification results of Fig. 2a–f. d, Robust cis P-tau signals are detected in neurons 48 h after rmTBI without any other tangle-related tau epitopes. 48 h after rmTBI, brain sections were stained with cis mAb (red) and AT8, AT100 or PHF1 (green). e, Robust cis P-tau signals are detected in neurons 48 h after rmTBI without tau oligomerization, which appear and colocalize with cis P-tau at 6 months after TBI. 48 h or 6 months after rmTBI or sham treatment, brain sections were immunostained with T22 (green) and cis or trans mAb (red). The results in 48 h sham mice were similar to those at 6 months (data not shown). The colocalization of red and green signals was quantified using Coloc-2, with the results being shown in percentages. ND, not detectable. n = 3–4. The results are expressed as means ± s.d. and P values determined using the Student’s t-test.
Extended Data Figure 4 Stressed neurons robustly produce cis P-tau, cis P-tau is released from stressed neurons and neurotoxic, but is effectively blocked by cis, but not trans, mAb.
a–c, Quantification results of Fig. 4a, b and f, respectively. The results are expressed as means ± s.d. and P values determined using the two-way ANOVA test (a) and Student’s t-test (c). d, Hypoxia induces cis P-tau, which is blocked by cis mAb. SY5Y neurons expressing a control vector were cultured in the hypoxia chamber in the absence or presence of cis or trans mAb for the times indicated, followed by immunoblotting for cis P-tau. e, Hypoxia induces cis P-tau before tau aggregation. SY5Y neurons were subjected to hypoxia for the times indicated, followed by sarkosyl extraction before immunoblotting with Tau5 mAb and quantification. f, Hypoxia induces cell death, which are blocked by cis, but not trans, mAb. SY5Y neurons were cultured in the hypoxia chamber in the absence or presence of cis or trans mAb for the times indicated, followed by live and dead cell assay using the LIVE/DEAD Viability/Cytotoxicity Kit. g, Stressed neuron lysates are neurotoxic, which are neutralized by cis, but not trans, mAb. Cell lysates were prepared from stressed SY5Y neurons and then added to growing SY5Y neurons directly (Control) or after immunodepletion with cis or trans mAb to remove cis or trans P-tau, respectively for 3 days, followed by live and dead cell assay. h, Cis P-tau is released from stressed neurons. SY5Y neurons were cultured in the absence of serum for the times indicated and culture media were collected and centrifuged, followed by analysing the supernatants for cis and trans P-tau with actin as an indicator of cell lysis.
Extended Data Figure 5 Cis P-tau spreads after rmTBI or neuronal stress, and hypoxia induces cell death in primary neurons, which is blocked by cis mAb.
a, Cis P-tau spreads in the brain after rmTBI. Quantification results of Fig. 3c. b, Cis P-tau spreads after neuronal stress. GFP–tau or RFP–tau SY5Y neurons were co-cultured and subjected to hypoxia or control treatment in the presence or absence of cis or trans mAb for different times, followed by assaying cells expressing both GFP–tau and RFP–tau (arrows) to determine tau spreading among cells. The results are expressed as means ± s.d. and P values determined using the Student’s t-test. c, Cis mAb enters primary neurons. Primary neurons were established from mouse embryos and differentiated in vitro and cis mAb was added to culture media, followed by immunostaining with secondary antibodies. d, Hypoxia induces cell death in primary neurons, which is effectively blocked by cis mAb. Primary neurons were cultured in the hypoxia chamber in the absence or presence of cis mAb for 48 h, followed by live (green) and dead (red) cell assay using the LIVE/DEAD Viability/Cytotoxicity Kit.
Extended Data Figure 6 Pin1 inhibition by multiple mechanisms contributes to cis P-tau induction after neuron stress and TBI.
a, Pin1 is downregulated and correlates with cis P-tau induction after serum starvation. Cells were subjected to serum starvation for times indicated, followed by immunoblotting, with the right panel showing the correlation of Pin1 down regulation with cis P-tau induction from Fig. 4a. b, Pin1 is oxidized and correlates with cis P-tau induction after hypoxia. SY5Y cells were subjected to hypoxia for times indicated, followed by immunoblotting for C113 oxidized Pin1, with the right panel showing the correlation of Pin1 oxidization with cis P-tau induction from Extended Data Fig. 6d. c, Pin1 is inhibited in TBI mouse brains. Mouse brains 48 h after ssTBI were subjected to immunoblotting and quantification for Pin1 and S71 phosphorylated Pin1. d, Pin1 knockdown potentiates the ability of hypoxia to induce cis P-tau. Pin1-knockdown or vector control SY5Y cells were subjected to hypoxia treatment for the times indicated in the presence or absence of cis mAb, followed by immunoblotting and quantification for cis P-tau levels. The results are expressed as means ± s.d.
Extended Data Figure 7 Inhibition of FcγR binding blocks cis mAb from entering neurons and TRIM21 KD fully prevented cis antibody from ablating cis P-tau in neurons.
a–d, Inhibition of FcγR binding potently blocks cis mAb from entering neurons. Cis mAb was added to neurons in the absence or presence of a human FcγR-binding inhibitor, followed by detecting the binding of cis mAb to the cell surface by FACS (a), entry of cis mAb into cells by immunofluorescence (b), immunoblotting (c) and electron microscopy after immunogold labelling (d). The FcR binding inhibitor fully blocked cis mAb from binding to the cell surface and entering neurons. Electron microscopy showed that cis mAb bound to the cell surface and endocytic vesicles (red arrows). e, f, TRIM21 knockdown fully prevents cis antibody from ablating cis P-tau in neurons. TRIM21 was stably knocked down in SY5Y neuronal cells using a validated TRIM21 shRNA lentiviral vector and confirmed by real-time RT–PCR analysis of TRIM21 mRNA expression (e). TRIM21 knockdown or vector control SY5Y cells were subjected to hypoxia treatment in the presence or absence of cis mAb and/or 3-methyladenine, an autophagy inhibitor, followed by immunoblotting, followed by quantifying cis P-tau levels normalized actin levels (lower panel) (f). The results are expressed as means ± s.d.
Extended Data Figure 8 Cis pT231-tau is both necessary and sufficient for P-tau to induce neuronal cell death in vitro.
a, SY5Y cells were co-transfected with non-tagged indicated constructs in the absence and presence of cis mAb followed by immunoblotting with quantification on the right panel. b, SY5Y cells were co-transfected with GFP–tau, or GFP–tau(T231A) and p25/Ckd5 in the absence and presence of cis or trans mAb followed by live-cell confocal video (see Supplementary Videos 5, 6). Red arrows point to GFP–tau or–tau(T231A) expressing cells. The results are expressed as means ± s.d.
Extended Data Figure 9 Cis mAb effectively blocks cis P-tau induction and spread, tau aggregation, and restores neuronal ultrastructures, apoptosis and defective LTP after TBI.
a, Peripherally administrated cis and trans mAbs enter neurons in brains. 250 µg of biotinylated cis or trans mAb was injected intraperitoneally or intravenously into B6 mice, followed by detecting the biotinylated cis mAb in brains 3 days later. b, c, Cis mAb effectively blocks cis pT231-tau induction and apoptosis. ssTBI mice were randomly and blindly treated with cis mAb or IgG isotype control, i.c.v. (intracerebroventricular) 20 µg per mouse 15 min after injury, and then i.p. 200 µg every 4 days for 3 times, followed by subjecting brains to immunoblotting for cis P-tau (b) and PARP cleavage (c), with sham as controls. d–f, Cis mAb effectively blocks cis pT231-tau induction and spread, tau aggregation and restores neuronal ultrastructures. ssTBI mice in c–f received additional i.p. 200 µg per mouse 3 day before injury. d, Quantification of immunoblotting in Fig. 5a. e, Quantification of immunoblotting in Fig. 5b. f, Quantification of electron microscopy images in Fig. 5c. n = 3. The results are expressed as means ± s.d. and P values determined using Student’s t-test. g, Cis mAb treatment of ssTBI mice rescues defective LTP in the cortex. fEPSPs were recorded in the layer II/III by stimulating the vertical pathway (the layer V to II/III) in the cortex. Robust LTP was induced by 5 Hz theta-burst in the cortical slices of sham mice (n = 15 slices, 9 mice), but was deficient in the cortex of IgG-treated TBI mice (n = 9 slices, 5 mice). However, LTP magnitude was restored to the control level in cis mAb-treated TBI animals (n = 9 slices, 5 mice). The representative recordings were presented. h, No significant effects of cis pT231-tau mAb treatment on Morris Water Maze performance. 8 weeks after ssTBI, mice underwent Morris Water Maze (MWM) testing consisting of 4 acquisition trials (hidden platform) daily for 4 days (4 runs per trial), a probe trial, followed by a 3 reversal trials (hidden platform) daily for 3 days. Compared to sham mice, injured mice demonstrated increased latency to find the hidden platform in acquisition and reversal trials (P <0.001). There was no difference in injured cis mAb mice compared to injured IgG treated mice in acquisition trials (P = 0.5) or reversal trial (P = 0.9). For probe trials, injured mice performed similarly to sham mice (P = 0.7) and injured cis mAb treated mice performed similarly to injured IgG treated mice (P = 0.2). n = 4–7. The results are expressed as means ± s.e.m. and P values determined using ANOVA.
Extended Data Figure 10 Cis mAb treatment effectively restores risk-taking behaviour and prevents tauopathy development and spread as well as brain atrophy after TBI.
a–c, Cis mAb treatment effectively restores risk-taking behaviour 2 months after ssTBI. Video-tracking data of each of all mice shows that ssTBI mice treated with cis mAb (n = 7) spent similar and very little time in the open arm compared to sham mice (n = 4), but much less time than TBI mice treated with IgG2b (n = 7) (a). Cis mAb-treated ssTBI mice had similar performance to sham in travelling velocity, but IgG2b-treated ssTBI mice travelled a greater velocity in the open arm (b). All three groups travelled similar total distance (c). Results are expressed as mean ± S.E.M. and P values determined using the Student’s t-test. d–f, Cis mAb treatment effectively prevents tauopathy development and spread as well as brain atrophy 6 months after ssTBI. ssTBI mice were treated with cis mAb or IgG control for 2 weeks or 6 months, with sham mice as controls, followed by immunofluorescence with various tauopathy epitopes (d), with immunostaining fluorescence intensity in the cortex and hippocampus being quantified (e), or to NeuN immunostaining for determining the thickness of the cortex and white matter at 6 months after TBI (f). n = 4.
Supplementary information
Supplementary Information
This file contains Supplementary Table 1 and Source Data images. (PDF 7035 kb)
Mitochondrial movement in control PC12 cells
Differentiated PC12 cells under normal condition were stained with MitoTracker Green FM and subjected for live-cell confocal imaging captured fast and slow transport of mitochondria (green dots) in the anterograde and retrograde directions along neurites. (AVI 2560 kb)
Hypoxia stops fast, but not slow mitochondrial movement in PC12 cells.
Differentiated PC12 cells cultured in a hypoxia chamber were stained with MitoTracker Green FM and subjected for live-cell confocal imaging. Hypoxia treatment, however, almost completely stopped fast transport of mitochondria (green dots) in both directions, without obvious effects on slow mitochondrial movement in the same neurites. (AVI 1519 kb)
cis mAb rescues defective fast mitochondrial movement induced by hypoxia in PC12 cells
Differentiated PC12 cells cultured in a hypoxia chamber in the presence of cis mAb were stained with MitoTracker Green FM and subjected for live-cell confocal imaging. cis mAb treatment restored the defective transport of mitochondria (green dots) in both anterograde and retrograde directions along neurites. (AVI 1768 kb)
trans mAb even accelerates retraction of entire neurites induced by hypoxia in PC12 cells
Differentiated PC12 cells cultured in a hypoxia chamber in the presence of trans mAb were stained with MitoTracker Green FM and subjected for live-cell confocal imaging. trans mAb treatment even accelerated retraction of entire neurites. (AVI 2094 kb)
Overexpression of tau and p25/Cdk5 causes neuronal death
HS-SY5Y cells were cotransfected with GFP-tau and p25/Cdk5 and subjected for live-cell imaging with confocal microscopy. GFP-tau-overexpressing cells (green) died after around 62 hr of transfection while the untransfected cells (phase) remain survive over the observation time. (AVI 841 kb)
cis mAb rescues neuronal death induced by overexpression of tau and p25/Cdk5
HS-SY5Y cells were cotransfected with GFP-tau and p25/Cdk5 and treated with cis mAb and subjected for live-cell imaging with confocal microscopy. cis mAb treatment blocked cell death caused by GFP-tau+p25/Cdk5 overexpression. Both transfected (green) and untransfected cells (phase) survived over the observation time. (AVI 882 kb)
Sham mice stayed mainly in the two closed or “safe” arms in the elevated plus maze
Sham mice were subjected to the elevated plus maze 2 months after injury. The mice were placed at the decision zone and they could enter any of these four arms within 5-minute period of test time. A video-tracking system was used to record the move of the mice. Sham mice largely stayed in the two closed or “safe” arms, exhibiting minimal risk-taking behavior. (AVI 4948 kb)
IgG-treated ssTBI mice explore the two open or “aversive” arms, displaying “risk-taking” behavior in the elevated plus maze
ssTBI mice were subjected to the elevated plus maze 2 months after IgG treatment. The mice were placed at the decision zone and they could enter any of these four arms within 5-minute period of test time. A video-tracking system was used to record the move of the mice. IgG-treated ssTBI mice strikingly explored the two open or “aversive” arms, displaying “risk-taking” behavior. (AVI 4946 kb)
cis mAb-treated ssTBI mice stayed mainly in the two closed or “safe” arms in the elevated plus maze
ssTBI mice were subjected to the elevated plus maze 2 months after cis mAb treatment. The mice were placed at the decision zone and they could enter any of these four arms within 5-minute period of test time. A video-tracking system was used to record the move of the mice. cis mAb-treated mice largely stayed in the two closed or “safe” arms, exhibiting minimal risk-taking behavior, similar to sham mice. (AVI 4996 kb)
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Kondo, A., Shahpasand, K., Mannix, R. et al. Antibody against early driver of neurodegeneration cis P-tau blocks brain injury and tauopathy. Nature 523, 431–436 (2015). https://doi.org/10.1038/nature14658
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DOI: https://doi.org/10.1038/nature14658
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