Nora Bengoa-Vergniory1,2, Elisavet Velentza-Almpani3, Ana Maria Silva1,4, Connor Scott5, Mariana Vargas-Caballero6, Magdalena Sastre3, Richard Wade-Martins7,8, Javier Alegre-Abarrategui9. 1. Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK. 2. Oxford Parkinson's Disease Centre, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK. 3. Department of Brain Sciences, Imperial College London, Hammersmith Hospital, London, W12 0NN, UK. 4. Medical Research Council Centre for Molecular Bacteriology and Infection, Department of Infectious Disease, Imperial College London, Armstrong Road, London, SW7 2AZ, UK. 5. Nuffield Department of Clinical Neurosciences, University of Oxford, Level 1, West Wing, John Radcliffe Hospital, Oxford, OX3 9DU, UK. 6. School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK. 7. Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK. richard.wade-martins@dpag.ox.ac.uk. 8. Oxford Parkinson's Disease Centre, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK. richard.wade-martins@dpag.ox.ac.uk. 9. Department of Brain Sciences, Imperial College London, Hammersmith Hospital, London, W12 0NN, UK. j.alegre@imperial.ac.uk.
Abstract
BACKGROUND: Multimerization is a key process in prion-like disorders such as Alzheimer's disease (AD), since it is a requirement for self-templating tau and beta-amyloid amyloidogenesis. AT8-immunohistochemistry for hyperphosphorylated tau is currently used for the diagnosis and staging of tau pathology. Given that tau-tau interactions can occur in the absence of hyperphosphorylation or other post-translational modifications (PTMs), the direct visualization of tau multimerization could uncover early pathological tau multimers. METHODS: Here, we used bimolecular fluorescent complementation, rapamycin-dependent FKBP/FRB-tau interaction and transmission electron microscopy to prove the in vitro specificity of tau-proximity ligation assay (tau-PLA). We then analyzed MAPT KO and P301S transgenic mice, and human hippocampus and temporal isocortex of all Braak stages with tau-PLA and compared it with immunohistochemistry for the diagnostic antibody AT8, the early phosphorylation-dependent AT180, and the conformational-dependent antibody MC1. Finally, we performed proteinase-K treatment to infer the content of amyloidogenic beta-sheet fold. RESULTS: Our novel tau-proximity ligation assay (tau-PLA) directly visualized tau-tau interactions in situ, and exclusively recognized tau multimers but not monomers. It elicited no signal in MAPT KO mouse brains, but extensively labelled P301S transgenic mice and AD brain. Two groups of structures were detected, a previously unreported widespread small-sized diffuse pathology and large, neurofibrillary-like lesions. Tau-PLA-labelled diffuse pathology appeared from the earliest Braak stages, mostly unaccompanied by tangle-like tau-immunohistochemistry, being significantly more sensitive than any small-sized dot-/thread-like pathology labelled by AT180-, AT8- and MC1-immunohistochemistry in most regions quantified at stages 0-II. Tau-PLA-labelled diffuse pathology was extremely sensitive to Proteinase-K, in contrast to large lesions. CONCLUSIONS: Tau-PLA is the first method to directly visualize tau multimers both in vitro and in situ with high specificity. We find that tau multimerization appears extensively from the earliest presymptomatic Braak stages as a previously unreported type of diffuse pathology. Importantly, in our study multimerization is the earliest detectable molecular event of AD tau pathology. Our findings open a new window to the study of early tau pathology, with potential implications in early diagnosis and the design of therapeutic strategies.
BACKGROUND: Multimerization is a key process in prion-like disorders such as Alzheimer's disease (AD), since it is a requirement for self-templating tau and beta-amyloid amyloidogenesis. AT8-immunohistochemistry for hyperphosphorylated tau is currently used for the diagnosis and staging of tau pathology. Given that tau-tau interactions can occur in the absence of hyperphosphorylation or other post-translational modifications (PTMs), the direct visualization of tau multimerization could uncover early pathological tau multimers. METHODS: Here, we used bimolecular fluorescent complementation, rapamycin-dependent FKBP/FRB-tau interaction and transmission electron microscopy to prove the in vitro specificity of tau-proximity ligation assay (tau-PLA). We then analyzed MAPT KO and P301S transgenic mice, and human hippocampus and temporal isocortex of all Braak stages with tau-PLA and compared it with immunohistochemistry for the diagnostic antibody AT8, the early phosphorylation-dependent AT180, and the conformational-dependent antibody MC1. Finally, we performed proteinase-K treatment to infer the content of amyloidogenic beta-sheet fold. RESULTS: Our novel tau-proximity ligation assay (tau-PLA) directly visualized tau-tau interactions in situ, and exclusively recognized tau multimers but not monomers. It elicited no signal in MAPT KO mouse brains, but extensively labelled P301S transgenic mice and AD brain. Two groups of structures were detected, a previously unreported widespread small-sized diffuse pathology and large, neurofibrillary-like lesions. Tau-PLA-labelled diffuse pathology appeared from the earliest Braak stages, mostly unaccompanied by tangle-like tau-immunohistochemistry, being significantly more sensitive than any small-sized dot-/thread-like pathology labelled by AT180-, AT8- and MC1-immunohistochemistry in most regions quantified at stages 0-II. Tau-PLA-labelled diffuse pathology was extremely sensitive to Proteinase-K, in contrast to large lesions. CONCLUSIONS: Tau-PLA is the first method to directly visualize tau multimers both in vitro and in situ with high specificity. We find that tau multimerization appears extensively from the earliest presymptomatic Braak stages as a previously unreported type of diffuse pathology. Importantly, in our study multimerization is the earliest detectable molecular event of AD tau pathology. Our findings open a new window to the study of early tau pathology, with potential implications in early diagnosis and the design of therapeutic strategies.
Entities:
Keywords:
AT8; Aggregation; Alzheimer’s; Early pathology; Multimer; Phosphorylation; Proximity-ligation assay; Tau
Authors: Brandon B Holmes; Jennifer L Furman; Thomas E Mahan; Tritia R Yamasaki; Hilda Mirbaha; William C Eades; Larisa Belaygorod; Nigel J Cairns; David M Holtzman; Marc I Diamond Journal: Proc Natl Acad Sci U S A Date: 2014-09-26 Impact factor: 11.205
Authors: Sarah K Kaufman; David W Sanders; Talitha L Thomas; Allison J Ruchinskas; Jaime Vaquer-Alicea; Apurwa M Sharma; Timothy M Miller; Marc I Diamond Journal: Neuron Date: 2016-10-27 Impact factor: 17.173
Authors: T Gómez-Isla; R Hollister; H West; S Mui; J H Growdon; R C Petersen; J E Parisi; B T Hyman Journal: Ann Neurol Date: 1997-01 Impact factor: 10.422
Authors: M Fá; D Puzzo; R Piacentini; A Staniszewski; H Zhang; M A Baltrons; D D Li Puma; I Chatterjee; J Li; F Saeed; H L Berman; C Ripoli; W Gulisano; J Gonzalez; H Tian; J A Costa; P Lopez; E Davidowitz; W H Yu; V Haroutunian; L M Brown; A Palmeri; E M Sigurdsson; K E Duff; A F Teich; L S Honig; M Sierks; J G Moe; L D'Adamio; C Grassi; N M Kanaan; P E Fraser; O Arancio Journal: Sci Rep Date: 2016-01-20 Impact factor: 4.379
Authors: Anthony W P Fitzpatrick; Benjamin Falcon; Shaoda He; Alexey G Murzin; Garib Murshudov; Holly J Garringer; R Anthony Crowther; Bernardino Ghetti; Michel Goedert; Sjors H W Scheres Journal: Nature Date: 2017-07-05 Impact factor: 49.962
Authors: Evan Lester; Felicia K Ooi; Nadine Bakkar; Jacob Ayers; Amanda L Woerman; Joshua Wheeler; Robert Bowser; George A Carlson; Stanley B Prusiner; Roy Parker Journal: Neuron Date: 2021-04-12 Impact factor: 17.173