Refining a Mouse Model of Progressive Supranuclear Palsy Through Inoculation of Human Post-Mortem Brain-Derived Tau

Refining a Mouse Model of Progressive Supranuclear Palsy Through Inoculation of Human Post-Mortem Brain-Derived Tau | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Refining a Mouse Model of Progressive Supranuclear Palsy Through Inoculation of Human Post-Mortem Brain-Derived Tau SH Qamar, A Mao, R Ferry, S Thapa, P Singh, MC Tartaglia, MS Pollanen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7456824/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2026 Read the published version in BMC Research Notes → Version 1 posted 12 You are reading this latest preprint version Abstract Objective A major obstacle to developing effective therapies for Progressive Supranuclear Palsy (PSP), a uniformly fatal 4R tauopathy, is the absence of an animal model that faithfully reproduces the anatomical, cytopathological, and spatiotemporal progression of disease. Inoculation-based models, using human postmortem brain material bearing disease-specific proteopathic tau seeds, hold great translational potential for modeling tauopathies. Here we conducted key studies towards the development of an inoculation-based PSP model, using human postmortem brain to target three subcortical nuclei impacted in early disease. Results We evaluated the impact of five different PSP brain extracts on the extent and distribution of tau pathology following inoculation into 6hTau transgenic mice expressing all six isoforms of human tau. Our findings demonstrate that 2% sarkosyl-insoluble tau successfully recapitulates core cytopathological features of PSP when introduced into disease-relevant nuclei. However, we also identify a major limitation in the restricted yield of 2% sarkosyl-insoluble tau, which significantly impedes the scalability and reproducibility of this approach. We conclude that further progress will likely require alternative strategies to generate a stable and scalable source of tau proteopathic seeds, to support a robust and reproducible inoculation-based mouse model of PSP. Progressive Supranuclear Palsy Tau Human Postmortem Brain Intracerebral Inoculation Animal Model Tau pathology Figures Figure 1 Figure 2 Introduction Progressive Supranuclear Palsy (PSP), characterized by accumulation of four repeat (4R) tau in neurons and glia in distinct brain regions( 1 ), is a uniformly fatal neurodegenerative disease with no existing treatment. In early disease, the substantia nigra (SN) exhibits neurofibrillary tangles and threads, the globus pallidus (GP) neuronal pathology and oligodendroglial coiled bodies, and the caudate putamen (CPu) harbors pathognomonic tufted astrocytes( 1 ). The spatiotemporal spread of these three distinct cytopathologies throughout the brain occurs in six sequential stages( 1 , 2 ) driven by the self-propagating prion-like behaviour of misfolded tau( 3 ). As a primary tauopathy, with a rapid clinical progression and strong clinico-pathologic correlation, PSP has been described as a frontrunner in translational value amongst the tauopathies( 4 ). However, a critical barrier in the development of desperately needed treatments, is that no animal model recapitulates the anatomic and cytopathologic hallmarks, spatiotemporal spread of pathology and progressive neurodegeneration that define the disease( 5 ). Current animal models therefore offer poor translational value, contributing to the failure of clinical trials of potential disease modifying therapies( 6 ). Inoculation-based models, using human postmortem brain extracts bearing disease-specific proteopathic tau seeds, hold great translational potential for modeling tauopathies( 7 ). Indeed, these studies, leveraging the self-propagating ability of misfolded tau, have demonstrated the feasibly of replicating all three key PSP cytopathologies, as well as the propagation of pathology in mouse brain( 8 – 13 ). However, studies to date, employing a range of mouse lines and a variety of methods to extract tau from the human brain, have not focussed on the key subcortical nuclei affected in PSP( 8 – 13 ). As a critical step in the development of an animal model of PSP, here we compare the effects of five different tau extraction methods from human PSP brain (as well as one extraction method from human Alzheimer’s disease (AD) brain) inoculated in the SN, GP and CPu of 6hTau transgenic mice that express all six isoforms of human tau( 9 ) (Supplementary Fig. 1). Methods Characterisation of naïve 6hTau mice hT-PAC-N+/+;E10 + 14+/-;Mapt-/- (6htau) mice were obtained from the University of Pennsylvania and maintained as previously described( 9 ). N = 6 (3 males and 3 females per group), were aged to 3, 6 and 12 months old. Animals were administered sodium pentobarbital (40 mg/kg intraperitoneally). Once an appropriate surgical plane of anesthesia was achieved (as verified by a loss of toe pinch flexor response) animals were perfused transcardiacally with PBS. Following perfusion, the right hemispheres were fixed in 4% paraformaldehyde and paraffin embedded for histology. The left hemispheres were homogenized in 10% w/v PBS and centrifuged (10,000g, 10 minutes, 4°C) and the supernatants subjected to 4R-tau seed amplification assay (SAA). 4R-tau SAA SAA reactions were performed as previously described, with minor modifications( 14 ). All reagents were purchased from Sigma. 10µl of biological sample (1.5µg total protein) was added to wells containing 20µl of reaction buffer (0.1M PB, pH 8, 0.875M Na3Citrate, 45µM Poly-L-glutamic acid sodium salt), 10µl of 50µM ThT and 10µl of a mixture of 0.5 mg/ml of monomeric 4R-K18 and 0.25mg/ml of monomeric 3R-K19 (rPeptide). The plate was incubated at 37°C in a BMG FLUOstar Omega plate reader with cycles of 1 min shaking (500 rpm double orbital) and 1 min rest. ThT fluorescence (450 ± 10 nm excitation and 480 ± 10 nm emission, bottom read) were measured every 15 min for a period of 40h. Immunohistochemical Analysis of tau pathology 6µm sections were deparaffinized, rehydrated and stained for tau hyperphosphorylated at Ser202/Thr205 using AT8 (MN1020, Thermo Scientific) using an automated immunostainer (Dako, Agilent) according to manufacturer’s instructions. Sections were assessed for aberrant tau cytopathologies (neuronal, oligodendroglial, astrocytic) by a neuropathologist blinded to the animals’ treatment using a 4-point semiquantitative scale was applied: 0 = none, 1 = minimal, 2 = mild, 3 = moderate and 4 = severe( 15 ). Human PSP and AD Case Selection Human brain material was collected with informed consent (University Health Network Research Ethics Board, Protocol 20-5258). The frontal cortex of a 73-year-old male with a clinical diagnosis of PSP for 3 years prior to death was selected. Neuropathological examination confirmed a diagnosis of PSP and excluded AD, argyrophilic grain disease, limbic-predominant age-related TDP-43 encephalopathy and Lewy body disease. A comprehensive biochemical characterisation of this case, reported in our previous study as PSP#20( 16 ), revealed abundant high molecular weight tau species as well as hyperphosphorylated and oligomeric tau, and high tau seeding activity as determined using a 4R-tau SAA. For AD inoculations, a 75-year male was selected, with an 11-year history of dementia and neuropathologically confirmed AD, Braak NFT stage 6 with frequent neuritic plaques. Preparation of Human Brain Extracts: All extracts were prepared using previously published methods, for full methodological details please refer to supplemental materials. 10% weight/volume (w/v) brain lysate 100mg of human brain tissue was homogenized in 1ml PBS in a final concentration of 10% (w/v), containing protease and phosphatase inhibitors and stored at -80°C prior to use. PBS Soluble tau PBS soluble tau was prepared as previously described( 16 ) using 50mg of dissected human tissue. 0.1% Sarkosyl-Insoluble (SI) tau 0.1% SI tau was prepared as previously described( 11 ) using 1g of human brain tissue homogenized in 10 volumes (w/v) extraction buffer. 1% Sarkosyl-Insoluble tau 1% SI tau was prepared as previously described( 8 – 13 ) using 2g of human brain tissue homogenized in 9 volumes (w/v) of extraction buffer. 2% Sarkosyl-Insoluble tau 1% SI tau was prepared as previously described( 17 ) using 2g of human brain tissue homogenized in 10 volumes (w/v) suspension buffer. Preparation of lysates for inoculation Total Tau was quantified (ELISA, INNOTEST hTAU Ag catalog# 81580) and the yield calculated by [(total tau in ng/µl) x (final volume of tau extract in µl)/ (grams of human tissue)]. Prior to injection lysates were diluted to the required concentration and sonicated (Bioruptor Pico). Intracerebral injection 8-week-old 6htau mice (N = 2 per group) were injected (2.5 µl /site) using stereotaxic surgical technique at the following coordinates from bregma and skull surface (medial-lateral, anterior–posterior, dorsal-ventral in mm): CPu (+ 1.5, + 0.75, -3.0), GP (+ 1.8, -0.34, -4.0), SN (+ 1.4, -3.16, -4.6), hippocampus (+ 2.0, 2.5, -2.4) and overlying cortex (+ 2.0, -2.5, -1.4). Modified Bielschowsky stain 6µm tissue sections were deparaffinized, rehydrated and subject to the Bielschowsky Method - Sevier-Munger Modification. Results Extraction method affects the yield of tau from PSP post-mortem brain 10% w/v brain lysates, PBS soluble extracts( 16 ) as well as 0.1%( 11 ), 1%( 9 ) and 2%( 17 ) sarkosyl insoluble (SI) tau extracts were prepared from the frontal cortex of a 73-year-old male with a diagnosis of PSP (Supplementary Fig. 2). Quantification of the tau yield using an enzyme-linked immunosorbent assay (INNOTEST, Fujirebio) demonstrated that the 10% w/v brain lysate yielded the greatest amount of total tau per gram of human brain (45,720ng/g), followed by the PBS soluble extract (14,666ng/g). The addition of sarkosyl had a dramatic impact on the tau yield with 0.1% sarkosyl yielding 115ng/g, 1% sarkosyl 27.8ng/g and 2% sarkosyl 4.3ng/g (Supplementary Fig. 1) . 6hTau mice do not develop spontaneous tau pathology Importantly, we demonstrate that uninoculated 6hTau mice showed no evidence of 4R-tau seeding activity (Supplementary Fig. 3a) , or AT8 immunopositivity in the brain, up to 12 months of age (Supplementary Fig. 3b-f) , suggesting that, under the present conditions, 6hTau mice do not spontaneously develop detectable 4R-tau seeding capacity or deposition of hyperphosphorylated tau within the first year of life. Inoculation with 2% SI human-brain derived PSP tau induces hyperphosphorylated tau deposits in 6hTau mice Next, 3-month-old 6hTau mice were inoculated in the CPu, SN and GP with 1ng tau per site (diluted in 2ul sterile PBS) from each of the 5 extracts, as well as PBS. Immunopositivity for hyperphosphorylated tau (AT8) was examined throughout the brain at 3- and 6-months post inoculation (mpi). Animals inoculated with PBS, as well as PBS soluble tau had little or no evidence of AT8 immunopositivity in the brain at 3 and 6 mpi ( Fig. 1 , Table 1). Animals inoculated with 10%w/v brain lysate, as well as 0.1% and 1% SI tau exhibited a moderate amount of AT8 immunopositivity at 6mpi with some spread to the contralateral hemisphere ( Fig. 1 , Table 1). In contrast, 2% SI PSP tau induced numerous AT8-positive tau deposits in neurons and neuropil threads proximal to the three inoculation sites 3 and 6 mpi, with the strongest pathology apparent in the SN. Furthermore, spread of neuronal pathology to anatomically connected regions, particularly the thalamus and hypothalamus, as well as the contralateral hemisphere was apparent at 6mpi. (Fig. 1 , Table 1 ). Glial pathology was scant, with only a single AT8 positive oligodendrocyte apparent in an animal inoculated with 0.1% SI tau, and a total of 3 tau positive astrocytes found across animals inoculated with 10%w/v brain lysate, 0.1% and 2% SI tau (Fig. 1 , Table 1 ). Inoculation with human-brain derived AD tau induces argentophilic tau deposits in 6hTau mice A separate group of 6hTau mice were inoculated in the CPu, SN and GP with 1% SI tau from a 75-year-old male with a diagnosis of AD. At 3 and 6mpi we found robust, exclusively neuronal, AT8 immunopositivity proximal to the inoculation sites with spread to several distal anatomically connected regions ( Supplementary Fig. 4 ). Finally, we examined the argentophilic properties of tau in both PSP and AD inoculated animals. Abundant argentophilic neurofibrillary tangles and neuropil threads were observed in the hippocampus of AD tau-inoculated mice, indicating the aggregation of misfolded tau proteins in neurons and dendrites. (Fig. 2 a, b). However, no silver positive neurofibrillary structures were evident in the brains of PSP inoculated animals (Fig. 2 c, d). Discussion Importantly, we demonstrate that 2% SI tau is the optimal extract to induce tau pathology upon inoculation, and that regions implicated in the early stages of PSP are capable of reproducing tau pathology, providing support for the use of this paradigm to model PSP. However, most tau deposits were neuronal, with only scant glial pathology apparent, and critically we did not observe the same distribution patterns of glial and neuronal pathology described in the CPu, GP and SN in PSP( 1 ). Furthermore, the yield of tau from PSP post-mortem brain decreased as increasing concentrations of sarkosyl were used, such that 1g of human PSP frontal cortex yielded sufficient 2% SI tau to induce only a modest tau pathology in the CPu, GP and SN of a single animal. Clearly this limited yield of proteopathic tau seeds from PSP post-mortem brain renders large-scale studies making direct use of patient-derived tau unfeasible. Although not explicitly stated, our findings are echoed in the literature, with studies using several grams of human brain, or pooling brain material from different patients, to obtain sufficient tau to inoculate only a modest number of animals (2–6 per study)( 8 – 13 ). Consistent with previous reports, that the yield of insoluble tau from PSP brain is lower than that of other tauopathies( 12 ). We found the yield of tau from AD brain was approximately double that of PSP. Furthermore, AD inoculated animals developed a robust, widespread AT8 and silver positive tau pathology, which provides a closer approximation of end-stage human pathology. Unlike AD-inoculated animals, PSP-inoculated mice failed to develop any neurofibrillary inclusions at the timepoints examined, suggesting that the deposition of hyperphosphorylated tau may occur independently of neurofibrillary tau formations and that tau hyperphosphorylation may be a preceding event to neurofibrillary tangle (NFT) development. Limitations Together our findings suggest that the exciting promise of using human brain derived tau to model PSP tau pathology in animals may be significantly hampered by the small quantity of seeding competent tau that can be directly extracted from human brain. Indeed this limits the number of animals inoculated in the present study and in published works ( 8 – 13 ). This situation is further compounded by the fact that PSP is a rare and heterogenous disease, thus only limited groups have access to postmortem material from well characterized cases. For preclinical drug development, the measurement of the effect size of disease modifying therapies will likely require an animal model that develops a substantial pathological burden. Thus, to progress, the field should consider new avenues such as employing in vitro seeding amplification reactions( 12 ), or cell-based approaches( 18 ) to generate the reliable, reproducible , and sustainable source of PSP proteopathic seeds that are essential for the generation of a robust, replicable and scalable mouse model of PSP. Abbreviations Progressive Supranuclear Palsy (PSP) four repeat (4R) substantia nigra (SN) globus pallidus (GP) caudate putamen (CPu) Alzheimer’s disease (AD) hT-PAC-N+/+;E10+14+/-;Mapt-/- (6htau) weight/volume (w/v) Sarkosyl-Insoluble (SI) months post inoculation (mpi) neurofibrillary tangle (NFT) Declarations Ethics approval and consent to participate Autopsy tissue from human brains were collected with informed consent of patients or their relatives and approval of the University Health Network Research Ethics Board (Nr. 20–5258). All animal use was in accordance with approved University Health Network, Animal Care Committee local protocol AUP 6556 and the regulations defined by the Canadian Council on Animal Care. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was funded by the Rossy Foundation, Blidner Family as well as grants from CurePSP and Brain Canada (NPV) and Canada Graduate Scholarship - the Canadian Institutes of Health Research, CRND Graduate Student Aid Endowment University of Toronto, URSO Student Fellowship CurePSP, University of Toronto Fellowships (SHQ). Supplementary Figure 1 was created in BioRender. Visanji, N. (2025) https://BioRender.com/tj7ylfv Authors' contributions SHQ Assisted with tau extractions, performed all immunohistochemistry and data analysis, and was a major contributor in writing the manuscript. AM performed tau extractions. RF was responsible for animal husbandry, breeding, genotyping and performing stereotaxic surgeries. ST performed the human total tau ELISA. PS performed sectioning of mouse brains. MCT oversaw the human total tau ELISA. AEL was involved in the conceptualisation of the study. HT performed the histological rating of Tau pathology. IM-V performed tau extractions and RTQuIC. MSP assisted in the execution and interpretation of silver staining. NPV Conceptualised and oversaw the study and was a major contributor in writing the manuscript. All authors read and approved the final manuscript. Acknowledgements We would like to thank the patients and family members who made the ultimate gift in donating their brains for this research. We are grateful to Dr Gerard Schellenberg from the University of Pennsylvania for provision of the animals used in this study. References Kovacs GG, Lukic MJ, Irwin DJ, Arzberger T, Respondek G, Lee EB, et al. Distribution patterns of tau pathology in progressive supranuclear palsy. Acta Neuropathol. 2020;140(2):99–119. Stamelou M, Respondek G, Giagkou N, Whitwell JL, Kovacs GG, Höglinger GU. Evolving concepts in progressive supranuclear palsy and other 4-repeat tauopathies. 17, Nat Reviews Neurol. 2021. Ayers JI, Paras NA, Prusiner SB. Expanding spectrum of prion diseases. Emerg Top Life Sci [Internet]. 2020 Sep 1 [cited 2022 Feb 6];4(2):155–67. Available from: https://pubmed.ncbi.nlm.nih.gov/32803268/ Shoeibi A, Olfati N, Litvan I. Frontrunner in translation: Progressive supranuclear palsy. Frontiers in Neurology. Volume 10. Frontiers Media S.A.; 2019. Priyanka, Qamar SH, Visanji NP. Toward an animal model of Progressive Supranuclear Palsy. Frontiers in Neuroscience. Volume 18. Frontiers Media SA; 2024. Vaswani PA, Olsen AL. Immunotherapy in progressive supranuclear palsy. Curr Opin Neurol [Internet]. 2020 Aug 1 [cited 2022 Feb 15];33(4):527–33. Available from: https://pubmed.ncbi.nlm.nih.gov/32657895/ Basheer N, Buee L, Brion JP, Smolek T, Muhammadi MK, Hritz J, et al. Shaping the future of preclinical development of successful disease-modifying drugs against Alzheimer’s disease: a systematic review of tau propagation models. Acta Neuropathologica Communications. Volume 12. 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Acta Neuropathol [Internet]. 2021;141:193–215. Available from: https://doi.org/10.1007/s00401-020-02253-4 Narasimhan S, Changolkar L, Riddle DM, Kats A, Stieber A, Weitzman SA et al. Human tau pathology transmits glial tau aggregates in the absence of neuronal tau. J Exp Med. 2020;217(2). Martinez-Valbuena I, Tartaglia MC, Fox SH, Lang AE, Kovacs GG. Four-Repeat Tau Seeding in the Skin of Patients With Progressive Supranuclear Palsy. JAMA Neurol [Internet]. 2024; Available from: http://www.ncbi.nlm.nih.gov/pubmed/39283648 Couto B, Martinez-Valbuena I, Lee S, Alfradique-Dunham I, Perrin RJ, Perlmutter JS, et al. Protracted course progressive supranuclear palsy. Eur J Neurol. 2022;29(8):2220–31. Martinez-Valbuena I, Lee S, Santamaria E, Fernandez Irigoyen J, Li J, Tanaka H et al. 4R-Tau seeding activity unravels molecular subtypes in patients with Progressive Supranuclear Palsy 2 3 4. Available from: https://doi.org/10.1101/2023.09.28.559953 Shi Y, Zhang W, Yang Y, Murzin AG, Falcon B, Kotecha A, et al. Structure-based classification of tauopathies. Nature. 2021;598(7880):359–63. Zeng Z, Vijayan V, Tsay K, Frost MP, Quddus A, Albert A et al. Springer Nature 2021 L A T E X template CBD and PSP cell-passaged Tau Seeds Generate Heterogeneous Fibrils with A sub-population Adopting Disease Folds. Available from: https://doi.org/10.1101/2023.07.19.549721 Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files QamaretalBMCsupplemetalmaterial.docx Table.png Table 1: Semiquantitative Neuropathological scoring of AT8 immunopositivity in 6hTau mice 3 and 6 months post inoculation (mpi) with PSP tau. 0=none, 1=minimal, 2= mild, 3=moderate, 4=severe. * represents injection sites. SupplementaryFigures.pdf Supplementary Figure 1: Schematic representation of PSP tau inoculation studies performed in 6htau mice. 1. The frontal cortex was obtained from a PSP postmortem brain. 2. Five different brain lysates were prepared. 3. Total tau was quantified in all lysates using an ELISA. 4. The yield of tau per gram of human brain was calculated for each of the lysates. 5. 6hTau mice were each inoculated in three subcortical nuclei implicated in early PSP. 6. Animal brains were examined for the presence of hyperphosphorylated tau (AT8 immunohistochemistry) and argentophilic neurofibrillary structures (modified Bielschowsy stain) at 3 and 6 months post inoculation. Supplementary Figure 2: AT8 immunostaining in the frontal cortex of the PSP case used in the present study. AT8 immunostaining reveals abundant neuronal pathology (closed arrowhead) tufted astrocytes (open arrowhead) and oligodendroglial coiled bodies (dashed arrowhead). Scale bar 200um. Supplementary Figure 3: Lack of 4R-tau seeding activity or AT8 immunopositivity in naive 6htau mouse brains at 3, 6 and 12 months of age. a) A 4R-Tau seeding amplification assay was performed in brain homogenates from a human PSP case, as well as 6hTau mice aged 3, 6 and 12 months of age. Immunostaining revealed a lack of AT8 immunopositivity throughout naïve 6hTau mouse brains at 3 (b), 6 (c) and 12 (d-f) months of age. Scale bar, show in f, represents 50um. Regions shown are caudate putamen (b-d), Globus pallidus (e) and substantia nigra pars compacta (f). Supplementary Figure 4: AT8 immunostaining in 6hTau mice 6 months post inoculation with AD tau. Animals were inoculated in the caudate putamen (CPu), Substantia Nigra pars compacta (SNc) and Globus Pallidus (GP) with 1% SI tau from an AD case. Images show ipsilateral hemisphere (a-c), and both ipsilateral (right side) and contralateral hemispheres (left side) in d. Scale bar shown in d: 100um (a-c), 300um (d). Table shows semiquantitative Neuropathological scoring of AT8 immunopositivity. 0=none, 1=minimal, 2= mild, 3=moderate, 4=severe. * represents injection sites. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7456824","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":511425296,"identity":"9e17c257-3e9e-438a-a1a3-93deeec62c1e","order_by":0,"name":"SH Qamar","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"SH","middleName":"","lastName":"Qamar","suffix":""},{"id":511425297,"identity":"bab9115e-f6f7-4701-9b68-f834db653905","order_by":1,"name":"A Mao","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"A","middleName":"","lastName":"Mao","suffix":""},{"id":511425298,"identity":"8dcaea05-c384-4ccb-ba55-5d4c3a8dd793","order_by":2,"name":"R Ferry","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"R","middleName":"","lastName":"Ferry","suffix":""},{"id":511425299,"identity":"e4e94489-4254-4388-8b43-a72e540e513e","order_by":3,"name":"S Thapa","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"S","middleName":"","lastName":"Thapa","suffix":""},{"id":511425300,"identity":"5d2e09f6-05e3-4c63-ae52-dbd9b86c4ebf","order_by":4,"name":"P Singh","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"P","middleName":"","lastName":"Singh","suffix":""},{"id":511425301,"identity":"46699127-7926-47c0-b21f-0ee31110fa2d","order_by":5,"name":"MC Tartaglia","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"MC","middleName":"","lastName":"Tartaglia","suffix":""},{"id":511425302,"identity":"6edc93c0-3dec-461e-874f-57c54464ec4f","order_by":6,"name":"MS Pollanen","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"MS","middleName":"","lastName":"Pollanen","suffix":""},{"id":511425303,"identity":"c7921c4c-68e2-4457-ae00-21f2a72cf05c","order_by":7,"name":"AE Lang","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"AE","middleName":"","lastName":"Lang","suffix":""},{"id":511425304,"identity":"d2fc97ce-dbca-4e15-bd7e-ab34dbcda567","order_by":8,"name":"H Tanaka","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"H","middleName":"","lastName":"Tanaka","suffix":""},{"id":511425305,"identity":"3d1bcb16-bb01-4a25-92ac-58a2d4d3497f","order_by":9,"name":"I Martinez-Valbuena","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYBACNjiLGURUkKKFB6zlDIMEmHeAGOt4QARjGxFa+CSSnz1gzLHLt2fnPfjw57zDdfzSh489/lDBIM/fgMNhEmnmBozbki17mPmSDSS3HZaQ7EtLNzhwhsFwBg6r2HgOmEkwbmM24GHmMZMwBGoxOANkHGxjSMDlOjae49+AWupBWsx/JM5B0iKPSwt7D8iWw2BbGA42IGkxwK2lTCJx23EDnsM8xpINx9IlZ/awpUmcOSNhuBGHFvlm9m0SH7dVG7D3nzH8+KPGmp+fh/mYREWFjbwcDi1gkIBFTAKP+lEwCkbBKBgFhAAAc01MRlbRU5IAAAAASUVORK5CYII=","orcid":"","institution":"University of Toronto","correspondingAuthor":true,"prefix":"","firstName":"I","middleName":"","lastName":"Martinez-Valbuena","suffix":""},{"id":511425306,"identity":"9c56e53f-6978-499c-ae75-fe1a24fd6a04","order_by":10,"name":"NP Visanji","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"NP","middleName":"","lastName":"Visanji","suffix":""}],"badges":[],"createdAt":"2025-08-25 20:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7456824/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7456824/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13104-025-07599-0","type":"published","date":"2026-01-06T15:59:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91186382,"identity":"9b8a535d-db06-487c-b124-0040ca65e3f1","added_by":"auto","created_at":"2025-09-12 13:58:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3165792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAT8 immunostaining in 6hTau mice 6 months post inoculation with PSP tau. \u003c/strong\u003eAnimals were inoculated in the caudate putamen (CPu), Globus Pallidus (GP) and Substantia Nigra pars compacta (SNc) with PBS (a-c), 10%w/v brain lysate (d-f), PBS soluble tau (g-i), 0.1% Sarkosyl Insoluble (SI) tau (j-l), 1% SI tau (m-o), 2% SI tau (p-u). Images show ipsilateral hemisphere. Scale bar shown in u represents: 100um (a-c), 20um (d-u).\u003c/p\u003e","description":"","filename":"QamaretalBMCFigures1.png","url":"https://assets-eu.researchsquare.com/files/rs-7456824/v1/6bd5661e33fed92e2faa81f8.png"},{"id":91185216,"identity":"fee7b98a-0b62-4b10-beac-ba3465d0b071","added_by":"auto","created_at":"2025-09-12 13:50:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1310207,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClassical neurofibrillary tangles and neuropil threads are present in AD tau- but not PSP tau-inoculated 6hTau mice. \u003c/strong\u003eStaining with the modified Bielschowsky method at a) 3 months post inoculation and b) 6 months post inoculation with AD 1% SI tau in the CPu, GP and SN. Mice inoculated with PSP 2% SI tau in the CPu, GP and SN c) 3 months post inoculation or d) 6 months post inoculation. Scale bar, shown in d represents 50um.\u003c/p\u003e","description":"","filename":"QamaretalBMCFigures2.png","url":"https://assets-eu.researchsquare.com/files/rs-7456824/v1/774742a6b9fa92fd5dcf9181.png"},{"id":100070100,"identity":"771adbd6-722c-46e4-8015-36487bc9a913","added_by":"auto","created_at":"2026-01-12 16:16:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5985195,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7456824/v1/3d4cf455-e5ad-413f-bd93-c8dd507fd41b.pdf"},{"id":91185218,"identity":"4ba22f2f-7645-4817-bfbb-7378b1599c8b","added_by":"auto","created_at":"2025-09-12 13:50:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":155385,"visible":true,"origin":"","legend":"","description":"","filename":"QamaretalBMCsupplemetalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7456824/v1/9eeb03c569f8e64bc6ba24c2.docx"},{"id":91186637,"identity":"62aee1e9-9bc7-42e7-bd82-0e34d4e9f158","added_by":"auto","created_at":"2025-09-12 14:06:15","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":192872,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1: Semiquantitative Neuropathological scoring of AT8 immunopositivity in 6hTau mice 3 and 6 months post inoculation (mpi) with PSP tau\u003c/strong\u003e. 0=none, 1=minimal, 2= mild, 3=moderate, 4=severe. * represents injection sites.\u003c/p\u003e","description":"","filename":"Table.png","url":"https://assets-eu.researchsquare.com/files/rs-7456824/v1/5d48f1517e2ef39f6a50b27b.png"},{"id":91186383,"identity":"38edb67b-f1c2-4c18-9d41-b921a43352c1","added_by":"auto","created_at":"2025-09-12 13:58:11","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1071333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1: Schematic representation of PSP tau inoculation studies performed in 6htau mice. \u003c/strong\u003e1. The frontal cortex was obtained from a PSP postmortem brain. 2. Five different brain lysates were prepared. 3. Total tau was quantified in all lysates using an ELISA. 4. The yield of tau per gram of human brain was calculated for each of the lysates. 5. 6hTau mice were each inoculated in three subcortical nuclei implicated in early PSP. 6. Animal brains were examined for the presence of hyperphosphorylated tau (AT8 immunohistochemistry) and argentophilic neurofibrillary structures (modified Bielschowsy stain) at 3 and 6 months post inoculation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2: AT8 immunostaining in the frontal cortex of the PSP case used in the present study. \u003c/strong\u003eAT8 immunostaining reveals abundant neuronal pathology (closed arrowhead) tufted astrocytes (open arrowhead) and oligodendroglial coiled bodies (dashed arrowhead). Scale bar 200um.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3: Lack of 4R-tau seeding activity or AT8 immunopositivity in naive 6htau mouse brains at 3, 6 and 12 months of age. \u003c/strong\u003ea) A 4R-Tau seeding amplification assay was performed in brain homogenates from a human PSP case, as well as 6hTau mice aged 3, 6 and 12 months of age. Immunostaining revealed a lack of AT8 immunopositivity throughout naïve 6hTau mouse brains at 3 (b), 6 (c) and 12 (d-f) months of age. Scale bar, show in f, represents 50um. Regions shown are caudate putamen (b-d), Globus pallidus (e) and substantia nigra pars compacta (f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4: AT8 immunostaining in 6hTau mice 6 months post inoculation with AD tau. \u003c/strong\u003eAnimals were inoculated in the caudate putamen (CPu), Substantia Nigra pars compacta (SNc) and Globus Pallidus (GP) with 1% SI tau from an AD case. Images show ipsilateral hemisphere (a-c), and both ipsilateral (right side) and contralateral hemispheres (left side) in d. Scale bar shown in d: 100um (a-c), 300um (d). Table shows semiquantitative Neuropathological scoring of AT8 immunopositivity. 0=none, 1=minimal, 2= mild, 3=moderate, 4=severe. * represents injection sites.\u003c/p\u003e","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7456824/v1/33c6c30936dd0183bfef5ec1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Refining a Mouse Model of Progressive Supranuclear Palsy Through Inoculation of Human Post-Mortem Brain-Derived Tau","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProgressive Supranuclear Palsy (PSP), characterized by accumulation of four repeat (4R) tau in neurons and glia in distinct brain regions(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), is a uniformly fatal neurodegenerative disease with no existing treatment. In early disease, the substantia nigra (SN) exhibits neurofibrillary tangles and threads, the globus pallidus (GP) neuronal pathology and oligodendroglial coiled bodies, and the caudate putamen (CPu) harbors pathognomonic tufted astrocytes(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The spatiotemporal spread of these three distinct cytopathologies throughout the brain occurs in six sequential stages(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) driven by the self-propagating prion-like behaviour of misfolded tau(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). As a primary tauopathy, with a rapid clinical progression and strong clinico-pathologic correlation, PSP has been described as a frontrunner in translational value amongst the tauopathies(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). However, a critical barrier in the development of desperately needed treatments, is that no animal model recapitulates the anatomic and cytopathologic hallmarks, spatiotemporal spread of pathology and progressive neurodegeneration that define the disease(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Current animal models therefore offer poor translational value, contributing to the failure of clinical trials of potential disease modifying therapies(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInoculation-based models, using human postmortem brain extracts bearing disease-specific proteopathic tau seeds, hold great translational potential for modeling tauopathies(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Indeed, these studies, leveraging the self-propagating ability of misfolded tau, have demonstrated the feasibly of replicating all three key PSP cytopathologies, as well as the propagation of pathology in mouse brain(\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). However, studies to date, employing a range of mouse lines and a variety of methods to extract tau from the human brain, have not focussed on the key subcortical nuclei affected in PSP(\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). As a critical step in the development of an animal model of PSP, here we compare the effects of five different tau extraction methods from human PSP brain (as well as one extraction method from human Alzheimer\u0026rsquo;s disease (AD) brain) inoculated in the SN, GP and CPu of 6hTau transgenic mice that express all six isoforms of human tau(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) \u003cb\u003e(Supplementary Fig.\u0026nbsp;1).\u003c/b\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCharacterisation of na\u0026iuml;ve 6hTau mice\u003c/h2\u003e\u003cp\u003ehT-PAC-N+/+;E10\u0026thinsp;+\u0026thinsp;14+/-;Mapt-/- (6htau) mice were obtained from the University of Pennsylvania and maintained as previously described(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). N\u0026thinsp;=\u0026thinsp;6 (3 males and 3 females per group), were aged to 3, 6 and 12 months old. Animals were administered sodium pentobarbital (40 mg/kg intraperitoneally). Once an appropriate surgical plane of anesthesia was achieved (as verified by a loss of toe pinch flexor response) animals were perfused transcardiacally with PBS. Following perfusion, the right hemispheres were fixed in 4% paraformaldehyde and paraffin embedded for histology. The left hemispheres were homogenized in 10% w/v PBS and centrifuged (10,000g, 10 minutes, 4\u0026deg;C) and the supernatants subjected to 4R-tau seed amplification assay (SAA).\u003c/p\u003e\u003cp\u003e\u003cb\u003e4R-tau SAA\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSAA reactions were performed as previously described, with minor modifications(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). All reagents were purchased from Sigma. 10\u0026micro;l of biological sample (1.5\u0026micro;g total protein) was added to wells containing 20\u0026micro;l of reaction buffer (0.1M PB, pH 8, 0.875M Na3Citrate, 45\u0026micro;M Poly-L-glutamic acid sodium salt), 10\u0026micro;l of 50\u0026micro;M ThT and 10\u0026micro;l of a mixture of 0.5 mg/ml of monomeric 4R-K18 and 0.25mg/ml of monomeric 3R-K19 (rPeptide). The plate was incubated at 37\u0026deg;C in a BMG FLUOstar Omega plate reader with cycles of 1 min shaking (500 rpm double orbital) and 1 min rest. ThT fluorescence (450\u0026thinsp;\u0026plusmn;\u0026thinsp;10 nm excitation and 480\u0026thinsp;\u0026plusmn;\u0026thinsp;10 nm emission, bottom read) were measured every 15 min for a period of 40h.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunohistochemical Analysis of tau pathology\u003c/h3\u003e\n\u003cp\u003e 6\u0026micro;m sections were deparaffinized, rehydrated and stained for tau hyperphosphorylated at Ser202/Thr205 using AT8 (MN1020, Thermo Scientific) using an automated immunostainer (Dako, Agilent) according to manufacturer\u0026rsquo;s instructions. Sections were assessed for aberrant tau cytopathologies (neuronal, oligodendroglial, astrocytic) by a neuropathologist blinded to the animals\u0026rsquo; treatment using a 4-point semiquantitative scale was applied: 0\u0026thinsp;=\u0026thinsp;none, 1\u0026thinsp;=\u0026thinsp;minimal, 2\u0026thinsp;=\u0026thinsp;mild, 3\u0026thinsp;=\u0026thinsp;moderate and 4\u0026thinsp;=\u0026thinsp;severe(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eHuman PSP and AD Case Selection\u003c/h3\u003e\n\u003cp\u003eHuman brain material was collected with informed consent (University Health Network Research Ethics Board, Protocol 20-5258). The frontal cortex of a 73-year-old male with a clinical diagnosis of PSP for 3 years prior to death was selected. Neuropathological examination confirmed a diagnosis of PSP and excluded AD, argyrophilic grain disease, limbic-predominant age-related TDP-43 encephalopathy and Lewy body disease. A comprehensive biochemical characterisation of this case, reported in our previous study as PSP#20(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), revealed abundant high molecular weight tau species as well as hyperphosphorylated and oligomeric tau, and high tau seeding activity as determined using a 4R-tau SAA. For AD inoculations, a 75-year male was selected, with an 11-year history of dementia and neuropathologically confirmed AD, Braak NFT stage 6 with frequent neuritic plaques.\u003c/p\u003e\n\u003ch3\u003ePreparation of Human Brain Extracts:\u003c/h3\u003e\n\u003cp\u003eAll extracts were prepared using previously published methods, for full methodological details please refer to supplemental materials.\u003c/p\u003e\u003cp\u003e\u003cb\u003e10% weight/volume (w/v) brain lysate\u003c/b\u003e\u003c/p\u003e\u003cp\u003e100mg of human brain tissue was homogenized in 1ml PBS in a final concentration of 10% (w/v), containing protease and phosphatase inhibitors and stored at -80\u0026deg;C prior to use.\u003c/p\u003e\n\u003ch3\u003ePBS Soluble tau\u003c/h3\u003e\n\u003cp\u003ePBS soluble tau was prepared as previously described(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) using 50mg of dissected human tissue.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e0.1% Sarkosyl-Insoluble (SI) tau\u003c/h2\u003e\u003cp\u003e0.1% SI tau was prepared as previously described(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) using 1g of human brain tissue homogenized in 10 volumes (w/v) extraction buffer.\u003c/p\u003e\u003cp\u003e\u003cb\u003e1% Sarkosyl-Insoluble tau\u003c/b\u003e\u003c/p\u003e\u003cp\u003e1% SI tau was prepared as previously described(\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) using 2g of human brain tissue homogenized in 9 volumes (w/v) of extraction buffer.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2% Sarkosyl-Insoluble tau\u003c/b\u003e\u003c/p\u003e\u003cp\u003e1% SI tau was prepared as previously described(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) using 2g of human brain tissue homogenized in 10 volumes (w/v) suspension buffer.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePreparation of lysates for inoculation\u003c/h3\u003e\n\u003cp\u003eTotal Tau was quantified (ELISA, INNOTEST hTAU Ag catalog# 81580) and the yield calculated by [(total tau in ng/\u0026micro;l) x (final volume of tau extract in \u0026micro;l)/ (grams of human tissue)]. Prior to injection lysates were diluted to the required concentration and sonicated (Bioruptor Pico).\u003c/p\u003e\n\u003ch3\u003eIntracerebral injection\u003c/h3\u003e\n\u003cp\u003e8-week-old 6htau mice (N\u0026thinsp;=\u0026thinsp;2 per group) were injected (2.5 \u0026micro;l /site) using stereotaxic surgical technique at the following coordinates from bregma and skull surface (medial-lateral, anterior\u0026ndash;posterior, dorsal-ventral in mm): CPu (+\u0026thinsp;1.5, +\u0026thinsp;0.75, -3.0), GP (+\u0026thinsp;1.8, -0.34, -4.0), SN (+\u0026thinsp;1.4, -3.16, -4.6), hippocampus (+\u0026thinsp;2.0, 2.5, -2.4) and overlying cortex (+\u0026thinsp;2.0, -2.5, -1.4).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eModified Bielschowsky stain\u003c/h2\u003e\u003cp\u003e6\u0026micro;m tissue sections were deparaffinized, rehydrated and subject to the Bielschowsky Method - Sevier-Munger Modification.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eExtraction method affects the yield of tau from PSP post-mortem brain\u003c/h2\u003e\u003cp\u003e10% w/v brain lysates, PBS soluble extracts(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) as well as 0.1%(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), 1%(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) and 2%(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) sarkosyl insoluble (SI) tau extracts were prepared from the frontal cortex of a 73-year-old male with a diagnosis of PSP \u003cb\u003e(Supplementary Fig.\u0026nbsp;2).\u003c/b\u003e Quantification of the tau yield using an enzyme-linked immunosorbent assay (INNOTEST, Fujirebio) demonstrated that the 10% w/v brain lysate yielded the greatest amount of total tau per gram of human brain (45,720ng/g), followed by the PBS soluble extract (14,666ng/g). The addition of sarkosyl had a dramatic impact on the tau yield with 0.1% sarkosyl yielding 115ng/g, 1% sarkosyl 27.8ng/g and 2% sarkosyl 4.3ng/g \u003cb\u003e(Supplementary Fig.\u0026nbsp;1)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003e6hTau mice do not develop spontaneous tau pathology\u003c/b\u003e\u003c/p\u003e\u003cp\u003eImportantly, we demonstrate that uninoculated 6hTau mice showed no evidence of 4R-tau seeding activity \u003cb\u003e(Supplementary Fig.\u0026nbsp;3a)\u003c/b\u003e, or AT8 immunopositivity in the brain, up to 12 months of age \u003cb\u003e(Supplementary Fig.\u0026nbsp;3b-f)\u003c/b\u003e, suggesting that, under the present conditions, 6hTau mice do not spontaneously develop detectable 4R-tau seeding capacity or deposition of hyperphosphorylated tau within the first year of life.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eInoculation with 2% SI human-brain derived PSP tau induces hyperphosphorylated tau deposits in 6hTau mice\u003c/h2\u003e\u003cp\u003eNext, 3-month-old 6hTau mice were inoculated in the CPu, SN and GP with 1ng tau per site (diluted in 2ul sterile PBS) from each of the 5 extracts, as well as PBS. Immunopositivity for hyperphosphorylated tau (AT8) was examined throughout the brain at 3- and 6-months post inoculation (mpi). Animals inoculated with PBS, as well as PBS soluble tau had little or no evidence of AT8 immunopositivity in the brain at 3 and 6 mpi \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eTable\u0026nbsp;1).\u003c/b\u003e Animals inoculated with 10%w/v brain lysate, as well as 0.1% and 1% SI tau exhibited a moderate amount of AT8 immunopositivity at 6mpi with some spread to the contralateral hemisphere \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eTable\u0026nbsp;1).\u003c/b\u003e In contrast, 2% SI PSP tau induced numerous AT8-positive tau deposits in neurons and neuropil threads proximal to the three inoculation sites 3 and 6 mpi, with the strongest pathology apparent in the SN. Furthermore, spread of neuronal pathology to anatomically connected regions, particularly the thalamus and hypothalamus, as well as the contralateral hemisphere was apparent at 6mpi. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e). Glial pathology was scant, with only a single AT8 positive oligodendrocyte apparent in an animal inoculated with 0.1% SI tau, and a total of 3 tau positive astrocytes found across animals inoculated with 10%w/v brain lysate, 0.1% and 2% SI tau (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eInoculation with human-brain derived AD tau induces argentophilic tau deposits in 6hTau mice\u003c/h2\u003e\u003cp\u003eA separate group of 6hTau mice were inoculated in the CPu, SN and GP with 1% SI tau from a 75-year-old male with a diagnosis of AD. At 3 and 6mpi we found robust, exclusively neuronal, AT8 immunopositivity proximal to the inoculation sites with spread to several distal anatomically connected regions (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e). Finally, we examined the argentophilic properties of tau in both PSP and AD inoculated animals. Abundant argentophilic neurofibrillary tangles and neuropil threads were observed in the hippocampus of AD tau-inoculated mice, indicating the aggregation of misfolded tau proteins in neurons and dendrites. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). However, no silver positive neurofibrillary structures were evident in the brains of PSP inoculated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eImportantly, we demonstrate that 2% SI tau is the optimal extract to induce tau pathology upon inoculation, and that regions implicated in the early stages of PSP are capable of reproducing tau pathology, providing support for the use of this paradigm to model PSP. However, most tau deposits were neuronal, with only scant glial pathology apparent, and critically we did not observe the same distribution patterns of glial and neuronal pathology described in the CPu, GP and SN in PSP(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Furthermore, the yield of tau from PSP post-mortem brain decreased as increasing concentrations of sarkosyl were used, such that 1g of human PSP frontal cortex yielded sufficient 2% SI tau to induce only a modest tau pathology in the CPu, GP and SN of a single animal. Clearly this limited yield of proteopathic tau seeds from PSP post-mortem brain renders large-scale studies making direct use of patient-derived tau unfeasible. Although not explicitly stated, our findings are echoed in the literature, with studies using several grams of human brain, or pooling brain material from different patients, to obtain sufficient tau to inoculate only a modest number of animals (2\u0026ndash;6 per study)(\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eConsistent with previous reports, that the yield of insoluble tau from PSP brain is lower than that of other tauopathies(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). We found the yield of tau from AD brain was approximately double that of PSP. Furthermore, AD inoculated animals developed a robust, widespread AT8 and silver positive tau pathology, which provides a closer approximation of end-stage human pathology. Unlike AD-inoculated animals, PSP-inoculated mice failed to develop any neurofibrillary inclusions at the timepoints examined, suggesting that the deposition of hyperphosphorylated tau may occur independently of neurofibrillary tau formations and that tau hyperphosphorylation may be a preceding event to neurofibrillary tangle (NFT) development.\u003c/p\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eLimitations\u003c/h2\u003e\u003cp\u003eTogether our findings suggest that the exciting promise of using human brain derived tau to model PSP tau pathology in animals may be significantly hampered by the small quantity of seeding competent tau that can be directly extracted from human brain. Indeed this limits the number of animals inoculated in the present study and in published works (\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This situation is further compounded by the fact that PSP is a rare and heterogenous disease, thus only limited groups have access to postmortem material from well characterized cases. For preclinical drug development, the measurement of the effect size of disease modifying therapies will likely require an animal model that develops a substantial pathological burden. Thus, to progress, the field should consider new avenues such as employing \u003cem\u003ein vitro\u003c/em\u003e seeding amplification reactions(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), or cell-based approaches(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) to generate the \u003cem\u003ereliable, reproducible\u003c/em\u003e, and \u003cem\u003esustainable\u003c/em\u003e source of PSP proteopathic seeds that are essential for the generation of a \u003cem\u003erobust, replicable\u003c/em\u003e and \u003cem\u003escalable\u003c/em\u003e mouse model of PSP.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eProgressive Supranuclear Palsy (PSP)\u003c/p\u003e\n\u003cp\u003efour repeat (4R)\u003c/p\u003e\n\u003cp\u003esubstantia nigra (SN)\u003c/p\u003e\n\u003cp\u003eglobus pallidus (GP)\u003c/p\u003e\n\u003cp\u003ecaudate putamen (CPu)\u003c/p\u003e\n\u003cp\u003eAlzheimer’s disease (AD)\u003c/p\u003e\n\u003cp\u003ehT-PAC-N+/+;E10+14+/-;Mapt-/- (6htau)\u003c/p\u003e\n\u003cp\u003eweight/volume (w/v)\u003c/p\u003e\n\u003cp\u003eSarkosyl-Insoluble (SI)\u003c/p\u003e\n\u003cp\u003emonths post inoculation (mpi)\u003c/p\u003e\n\u003cp\u003eneurofibrillary tangle (NFT)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAutopsy tissue from human brains were collected with informed consent of patients or their relatives and approval of the University Health Network Research Ethics Board (Nr. 20\u0026ndash;5258). All animal use was in accordance with approved University Health Network, Animal Care Committee local protocol AUP 6556 and the regulations defined by the Canadian Council on Animal Care.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Rossy Foundation, Blidner Family as well as grants from CurePSP and Brain Canada (NPV) and Canada Graduate Scholarship - the Canadian Institutes of Health Research, CRND Graduate Student Aid Endowment University of Toronto, URSO Student Fellowship CurePSP, University of Toronto Fellowships (SHQ). \u0026nbsp;Supplementary Figure 1 was created in BioRender. Visanji, N. (2025) https://BioRender.com/tj7ylfv\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSHQ Assisted with tau extractions, performed all immunohistochemistry and data analysis, and was a major contributor in writing the manuscript.\u003c/p\u003e\n\u003cp\u003eAM performed tau extractions.\u003c/p\u003e\n\u003cp\u003eRF was responsible for animal husbandry, breeding, genotyping and performing stereotaxic surgeries.\u003c/p\u003e\n\u003cp\u003eST performed the human total tau ELISA.\u003c/p\u003e\n\u003cp\u003ePS performed sectioning of mouse brains.\u003c/p\u003e\n\u003cp\u003eMCT oversaw the human total tau ELISA.\u003c/p\u003e\n\u003cp\u003eAEL was involved in the conceptualisation of the study.\u003c/p\u003e\n\u003cp\u003eHT performed the histological rating of Tau pathology.\u003c/p\u003e\n\u003cp\u003eIM-V performed tau extractions and RTQuIC.\u003c/p\u003e\n\u003cp\u003eMSP assisted in the execution and interpretation of silver staining.\u003c/p\u003e\n\u003cp\u003eNPV Conceptualised and oversaw the study and was a major contributor in writing the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the patients and family members who made the ultimate gift in donating their brains for this research. We are grateful to Dr Gerard Schellenberg from the University of Pennsylvania for provision of the animals used in this study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKovacs GG, Lukic MJ, Irwin DJ, Arzberger T, Respondek G, Lee EB, et al. Distribution patterns of tau pathology in progressive supranuclear palsy. Acta Neuropathol. 2020;140(2):99\u0026ndash;119.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStamelou M, Respondek G, Giagkou N, Whitwell JL, Kovacs GG, H\u0026ouml;glinger GU. Evolving concepts in progressive supranuclear palsy and other 4-repeat tauopathies. 17, Nat Reviews Neurol. 2021.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAyers JI, Paras NA, Prusiner SB. Expanding spectrum of prion diseases. Emerg Top Life Sci [Internet]. 2020 Sep 1 [cited 2022 Feb 6];4(2):155\u0026ndash;67. 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Four-Repeat Tau Seeding in the Skin of Patients With Progressive Supranuclear Palsy. JAMA Neurol [Internet]. 2024; Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/39283648\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/pubmed/39283648\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCouto B, Martinez-Valbuena I, Lee S, Alfradique-Dunham I, Perrin RJ, Perlmutter JS, et al. Protracted course progressive supranuclear palsy. Eur J Neurol. 2022;29(8):2220\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartinez-Valbuena I, Lee S, Santamaria E, Fernandez Irigoyen J, Li J, Tanaka H et al. 4R-Tau seeding activity unravels molecular subtypes in patients with Progressive Supranuclear Palsy 2 3 4. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/2023.09.28.559953\u003c/span\u003e\u003cspan address=\"10.1101/2023.09.28.559953\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi Y, Zhang W, Yang Y, Murzin AG, Falcon B, Kotecha A, et al. Structure-based classification of tauopathies. Nature. 2021;598(7880):359\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZeng Z, Vijayan V, Tsay K, Frost MP, Quddus A, Albert A et al. Springer Nature 2021 L A T E X template CBD and PSP cell-passaged Tau Seeds Generate Heterogeneous Fibrils with A sub-population Adopting Disease Folds. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/2023.07.19.549721\u003c/span\u003e\u003cspan address=\"10.1101/2023.07.19.549721\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\n\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Progressive Supranuclear Palsy, Tau, Human Postmortem Brain, Intracerebral Inoculation, Animal Model, Tau pathology","lastPublishedDoi":"10.21203/rs.3.rs-7456824/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7456824/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eA major obstacle to developing effective therapies for Progressive Supranuclear Palsy (PSP), a uniformly fatal 4R tauopathy, is the absence of an animal model that faithfully reproduces the anatomical, cytopathological, and spatiotemporal progression of disease. Inoculation-based models, using human postmortem brain material bearing disease-specific proteopathic tau seeds, hold great translational potential for modeling tauopathies. Here we conducted key studies towards the development of an inoculation-based PSP model, using human postmortem brain to target three subcortical nuclei impacted in early disease.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe evaluated the impact of five different PSP brain extracts on the extent and distribution of tau pathology following inoculation into 6hTau transgenic mice expressing all six isoforms of human tau. Our findings demonstrate that 2% sarkosyl-insoluble tau successfully recapitulates core cytopathological features of PSP when introduced into disease-relevant nuclei. However, we also identify a major limitation in the restricted yield of 2% sarkosyl-insoluble tau, which significantly impedes the scalability and reproducibility of this approach. We conclude that further progress will likely require alternative strategies to generate a stable and scalable source of tau proteopathic seeds, to support a robust and reproducible inoculation-based mouse model of PSP.\u003c/p\u003e","manuscriptTitle":"Refining a Mouse Model of Progressive Supranuclear Palsy Through Inoculation of Human Post-Mortem Brain-Derived Tau","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 13:50:06","doi":"10.21203/rs.3.rs-7456824/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-15T15:36:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-06T06:23:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-05T22:40:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254209628346592730827673804867326235903","date":"2025-09-29T13:29:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79071958792979050718106835251140924536","date":"2025-09-26T03:06:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T09:27:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"231776815371390172673610324301630544830","date":"2025-09-12T16:10:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-07T14:04:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-07T14:01:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-05T09:47:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-04T16:52:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2025-09-04T16:47:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cf0ec26e-f8ac-4b1c-8398-8dab2b524ffa","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:09:58+00:00","versionOfRecord":{"articleIdentity":"rs-7456824","link":"https://doi.org/10.1186/s13104-025-07599-0","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2026-01-06 15:59:06","publishedOnDateReadable":"January 6th, 2026"},"versionCreatedAt":"2025-09-12 13:50:06","video":"","vorDoi":"10.1186/s13104-025-07599-0","vorDoiUrl":"https://doi.org/10.1186/s13104-025-07599-0","workflowStages":[]},"version":"v1","identity":"rs-7456824","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7456824","identity":"rs-7456824","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}