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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

72232
10.1038/s41598-024-72232-2
Article
The cellular prion protein does not affect tau seeding and spreading of sarkosyl-insoluble fractions from Alzheimer’s disease
Sala-Jarque Julia 123410
Gil Vanessa 1234
Andrés-Benito Pol 35
Martínez-Soria Inés 1234
Picón-Pagès Pol 1234
Hernández Félix 36
Ávila Jesús 36
Lanciego José Luis 37
Nuvolone Mario 9
Aguzzi Adriano 8
Gavín Rosalina 1234
Ferrer Isidro 35
del Río José Antonio jadelrio@ibecbarcelona.eu

1234
1 https://ror.org/056h71x09 grid.424736.0 0000 0004 0536 2369 Molecular and Cellular Neurobiotechnology, Institute for Bioengineering of Catalonia (IBEC), Parc Científic de Barcelona, Baldiri and Reixac 15-21, 08028 Barcelona, Spain
2 https://ror.org/021018s57 grid.5841.8 0000 0004 1937 0247 Department of Cell Biology, Physiology and Immunology, Faculty of Biology, University of Barcelona, Barcelona, Spain
3 https://ror.org/00ca2c886 grid.413448.e 0000 0000 9314 1427 Ciberned (Network Centre of Biomedical Research of Neurodegenerative Diseases), Institute of Health Carlos III, Madrid, Spain
4 https://ror.org/021018s57 grid.5841.8 0000 0004 1937 0247 Institute of Neuroscience, University of Barcelona, Barcelona, Spain
5 grid.411129.e 0000 0000 8836 0780 Department of Pathology and Experimental Therapeutics, University of Barcelona, Bellvitge University Hospital-IDIBELL, Hospitalet de Llobregat, Barcelona, Spain
6 grid.465524.4 Centro de Biología Molecular ‘Severo Ochoa’ (CBMSO) CSIC/UAM, Madrid, Spain
7 https://ror.org/02rxc7m23 grid.5924.a 0000 0004 1937 0271 Department of Neurosciences, Center for Applied Medical Research (CIMA), Universidad de Navarra, Pamplona, Spain
8 https://ror.org/00s6t1f81 grid.8982.b 0000 0004 1762 5736 Amyloidosis Research and Treatment Center, Foundation Scientific Institute Policlinico San Matteo, Department of Molecular Medicine, University of Pavia, Pavia, Italy
9 https://ror.org/02crff812 grid.7400.3 0000 0004 1937 0650 Institute of Neuropathology, University of Zurich, Zurich, Switzerland
10 https://ror.org/02t274463 grid.133342.4 0000 0004 1936 9676 Present Address: Neuroscience Research Institute and Molecular, Cell and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA USA
16 9 2024
16 9 2024
2024
14 2162230 3 2024
4 9 2024
© The Author(s) 2024
2024
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The cellular prion protein (PrPC) plays many roles in the developing and adult brain. In addition, PrPC binds to several amyloids in oligomeric and prefibrillar forms and may act as a putative receptor of abnormal misfolded protein species. The role of PrPC in tau seeding and spreading is not known. In the present study, we have inoculated well-characterized sarkosyl-insoluble fractions of sporadic Alzheimer’s disease (sAD) into the brain of adult wild-type mice (Prnp+/+), Prnp0/0 (ZH3 strain) mice, and mice over-expressing the secreted form of PrPC lacking their GPI anchor (Tg44 strain). Phospho-tau (ptau) seeding and spreading involving neurons and oligodendrocytes were observed three and six months after inoculation. 3Rtau and 4Rtau deposits from the host tau, as revealed by inoculating Mapt0/0 mice and by using specific anti-mouse and anti-human tau antibodies suggest modulation of exon 10 splicing of the host mouse Mapt gene elicited by exogenous sAD-tau. However, no tau seeding and spreading differences were observed among Prnp genotypes. Our results show that PrPC does not affect tau seeding and spreading in vivo.

Keywords

PrPC
Prnp
Tau
Mapt
Alzheimer’s disease
Seeding
Spreading
Subject terms

Neuroscience
Diseases of the nervous system
Neurodegeneration
http://dx.doi.org/10.13039/501100004837 Ministerio de Ciencia e Innovación PRPCDEVTAU PID2021-123714OB-I00 PRPCDEVTAU PID2021-123714OB-I00 PRE2022-103197 PRPCDEVTAU PID2021-123714OB-I00 PRPCDEVTAU PID2021-123714OB-I00 Sala-Jarque Julia Gil Vanessa Martínez-Soria Inés Gavín Rosalina del Río José Antonio http://dx.doi.org/10.13039/501100003030 Agència de Gestió d'Ajuts Universitaris i de Recerca SGR2021-00453 SGR2021-00453 SGR2021-00453 SGR2021-00453 Sala-Jarque Julia Gil Vanessa Gavín Rosalina del Río José Antonio http://dx.doi.org/10.13039/501100010805 Fundación Tatiana Pérez de Guzmán el Bueno http://dx.doi.org/10.13039/100010434 'la Caixa' Foundation LCF/PR/HR19/52160007 LCF/PR/HR19/52160007 LCF/PR/HR19/52160007 LCF/PR/HR19/52160007 LCF/PR/HR19/52160007 Gil Vanessa Andrés-Benito Pol Gavín Rosalina Ferrer Isidro del Río José Antonio Institute of Neuroscience, University of BarcelonaMDM-2017-0729 Gil Vanessa http://dx.doi.org/10.13039/501100015496 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Human tauopathies are clinically, neuropathologically, and biochemically distinct neurodegenerative diseases characterized by the deposition of abnormally misfolded and aggregated microtubule-associated protein tau in neurons and glial cells1–4. The principal tau deposits comprise 4Rtau, 3Rtau, or 3Rtau + 4Rtau, resulting from the alternative splicing of exon 10 of the tau gene Mapt5. Alzheimer’s disease is a combined 3R + 4R tauopathy and β-amyloidopathy6–8; frontotemporal lobar degeneration linked to MAPT mutations (FTLD-tau), argyrophilic grain disease (AGD), primary age-related tauopathy (PART), globular glial tauopathy (GGT), progressive supranuclear palsy (PSP), and age-related tau astrogliopathy (ARTAG) are 4R tauopathies and Pick’s disease is a 3R tauopathy3,9–17.

The neuropathology of tauopathies depends on several factors, including the genetic background, selective cellular and regional vulnerability, and progression. One of the mechanisms of disease progression is the transmission of abnormal proteins from one cell to another, reminiscent of prion transmission18. The hypothesis is supported by the capacity to seed an inoculated abnormal protein into the brain of a host and the spreading to connected brain regions19. Abnormal tau from tau-enriched brain homogenate fractions from distinct tauopathies has been used as inoculums in several mouse models, thus further validating the capacity of abnormal tau to seed, recruit host tau, transform host tau into an aberrant tau species, and transmit the new abnormal tau to neighboring cells. Mouse hosts include transgenic mice expressing human mutant tau19–22, mice over-expressing tau23, and wild type mice16,24–30.

Several mechanisms are implicated in the uptake of abnormal proteins. One of them is mediated by specific receptors. Focusing on tau, the low-density lipoprotein receptor-related protein 1 (LRP1)31,32, the cellular prion protein (PrPC)33–35, and the lymphocyte-activation gene 3 (LAG3)36,37 have been described as functional neuronal receptors of tau in experimental models.

PrPC is a cell surface GPI-anchored protein expressed in several tissues, with high levels expressed in neurons and glial cells38–41. The PrPC central region contains a central hydrophobic domain (HD or HR, aa 110/113–133) and a charged cluster domain (CD, aa 94–110), both involved in binding to distinct oligomeric/fibrillar amyloids, such as prions, β-amyloid and α-synuclein42–51. Over-expressed PrPC binds tau33–35, and PrPC mediates the hippocampal synaptic plasticity by soluble tau52. However, the putative role of cellular prion protein in tau seeding and spreading in wild-type mice is still unclear.

The present study is designed to assess the role of PrPC in tau seeding and spreading following the intra-cerebral inoculation of sarkosyl-insoluble fractions from sAD in mouse models. For this purpose, in addition to wild-type mice (Prnp+/+), we have used mutant Prnp mice over-expressing the GPI truncated form of the cellular prion protein: the “anchorless” GPI− Prnp (Tg44) mouse strain53 and ZH3 mouse strain lacking Prnp mice54.

Results

Characterization of the tau species used in the study

Western blotting of the sarkosyl-insoluble fractions of sAD cases was processed with the anti-ptau Ser422 (pSer422) antibody. The best profile was obtained in the case of a 65-year-old woman with dementia, showing three bands of around 68, 64, and 60 kDa, together with an upper band of 73 kDa, several bands of about 50 kDa, bands between 30 to 40 kDa, and two lower bands of truncated tau at the C-terminal, one of which was of about 25 kDa; a smear of higher molecular weight-represented large tau aggregates (Fig. 1A). TEM of the same sarkosyl-insoluble fraction revealed the presence of typical paired helical filaments (Fig. 1B). In parallel, we analyzed with TEM the aggregation of the tau K18 fragment in the presence (Hep +) or absence (Hep −) of heparin, to be used as controls in our in vitro experiments. As expected, K18 only forms fibers in the presence of heparin, as revealed in TEM (Fig. 1C,D). Next, we explored the seeding properties of the sarkosyl-insoluble fraction of the sAD patient using the Tau RD P301S Biosensor cell line. Tau K18 (Hep +) (Fig. 1F), sarkosyl-insoluble fractions of P301S transgenic mice (sP301S+/−) (Fig. 1I), and the sarkosyl-insoluble fraction of the sAD patient (Fig. 1J) were able to induce the formation of numerous eGFP aggregates in the cytoplasm of the cell line, thereby indicating their seeding properties. In contrast, no or very few eGFP aggregates were seen following incubation with tau K18 (Hep −) (Fig. 1G), and no aggregates were observed with the sarkosyl-insoluble fraction from P301S non-transgenic mice (sP301S−/−) (Fig. 1H), and the vehicle (Fig. 1E).Fig. 1 Characterization of the sarkosyl-insoluble fraction. (A) Western blot of the selected sarkosyl-insoluble fraction from the AD patient (sAD) immunoblotted using the pSer422 antibody showing three bands of about 68, 64, and 60 kDa, together with an upper band of 73 kDa, several bands of about 50 kDa, bands between 30 and 40 kDa, and two lower bands of truncated tau at the C-terminal. (B–D) TEM negative staining of the sAD sarkosyl-insoluble fractions (B) and tau K18 fragment after their fibrillation with (C) or without heparin (D). (E–J) seed competency experiments using the Tau RD P301S cell line, including vehicle (E), fibrillar tau K18 (F), non-fibrillated tau K18 (G), sarkosyl-insoluble fraction from a P301S non-transgenic mouse (H), P301S transgenic mouse (I), and sAD (J). Notice the presence of fluorescence aggregates in (F), (I), and (J) in contrast to vehicle, non-fibrillated tau K18, and sP301S−/− (wild-type) extracts. Scale bars: A–C = 0.5 µm, E–J = 200 µm; insert in J = 50 µm.

In additional controls, the treatment of the Tau RD P301S Biosensor cell line with (i) monomeric tau Cy5 (Supplementary Fig. S1A–C), (ii) murine or human preformed fibrils of α-synuclein (Supplementary Fig. S1D–E), and (iii) sarkosyl-insoluble brain extracts from multiple system atrophy (MSA) (Supplementary Fig. S1F), and Parkinson’s disease (PD) (Supplementary Fig. S1G) were unable to generate intracellular fluorescence aggregates. In contrast, treatments of Tau RD P301S cells with sarkosyl-insoluble fractions from aging-related tau astrogliopathy (ARTAG, a 4R tauopathy) (Supplementary Fig. S1H); Pick’s disease (PiD, a 3R tauopathy) (Supplementary Fig. S1I), and globular glial tauopathy (GGT, a 4R tauopathy) (Supplementary Fig. S1J) used in previous studies (Ferrer et al., 2018; Ferrer et al., 2022; Ferrer et al., 2020) render intracellular fluorescence aggregates indicating tau seeding properties.

Characterization of PrPC expression in mouse strains used in the study

The expression of PrPC was assessed in wild-type (Prnp+/+), ZH3 Prnp0/0, and GPI− Prnp mouse hippocampus using Western blot and immunohistochemistry using the 6H4 anti-PrPC antibody (Fig. 2, Supplementary Fig. S2). In parallel, PrPC was absent in ZH3 Prnp0/0 mouse extracts; a band of around 25kDa was obtained with GPI− Prnp brain extracts, as previously described53. In contrast, three bands were observed in (Prnp+/+) brain extracts (Fig. 2A). Next, sections from wild-type (Fig. 2B), ZH3 Prnp0/0 (Fig. 2C), and GPI– Prnp (Fig. 2D) mice were processed for PrPC immunohistochemistry. PrPC immunostaining was intense in Prnp+/+, particularly in the CA1 region, and lesser in the dentate gyrus (Fig. 2B). In contrast, the ZH3 Prnp0/0 (Fig. 2C) and GPI− Prnp (Fig. 2D) hippocampus showed a pale 6H4 labeling consistent with non-specific background. Lack of PrPC immunostaining was corroborated in hippocampal sections from Nestin-cre Prnpflox/flox mice compared to Prnpflox/flox lacking cre expression (Fig. 2E–F).Fig. 2 PrPC expression in Prnp+/+; ZH3 Prnp0/0, and GPI− Prnp mice. (A) Western blot of brain extracts from the three genotypes immunoblotted using 6H4 against PrPC and actin as a protein loading control. Note the absence of PrPC labeling in ZH3 Prnp0/0, the unique lower band in anchorless PrPC mice, and the triple band in wild-type mice Prnp+/+. (B–D) Low-power photomicrographs illustrating coronal hippocampal sections immunostained with anti-PrPC antibodies. Intense immunolabelling is seen in Prnp+/+ hippocampus, especially in the CA1 region (B). In contrast, a pale uniform background (negative immunostaining) is observed in the ZH3 Prnp0/0 and GPI− Prnp hippocampus (C–D). (E–F) 6H4 immunostaining in the Prnpflox/flox (E) Nestin-cre / Prnpflox/flox (F). Notice the absence of PrPC labelling in Nestin-cre/Prnpflox/flox hippocampus. CA1–CA3: cornus ammonis 1 and 3; DG: Dentate gyrus; H: Hilus; SO: stratum oriens; SP: stratum pyramidale; SR: stratum radiatum; IRL: interphase radiatum-lacunosum moleculare; SLM: stratum lacunosum-moleculare; ML: molecular layer; GCL: granule cell later. Scale bars: B–F = 500 µm.

The absence of PrPC expression or the expression of anchorless PrPC does not impair endogenous tau aggregation after sAD inoculation

ZH3 Prnp0/0, GPI− Prnp, and wild-type (Prnp+/+) mice aged 4–5 months were inoculated with sAD in the cortex/corpus callosum and hippocampus and analyzed 3 and 6 months later (Fig. 3). As controls, 4 wild-type mice were inoculated either with PBS 0.1M (n = 2), sarkosyl fractions of a non-AD patients (n = 2) and processed 3 months post-inoculation (Supplementary Fig. S3). Numerous AT8-positive deposits were found in sAD inoculated mice irrespective of their genotype and post-inoculation time (Fig. 3) in contrast to PBS 0.1M or non-AD sarkosyl fractions (Supplementary Fig. S3). AT8-positive inclusions of different morphologies (pre-tangles, threads, and granular, but not neurofibrillary tangles) were observed mainly in neurons (Fig. 3G), and oligodendrocytes (Fig. 3K–M) immunolabeled with AT8 and MC-1 (Fig. 3E–F) antibodies, but not in astrocytes (Fig. 3HI). Numerous AT8-positive ptau deposits were observed in axonal tracts (Fig. 3J). Ptau inclusions were also positive with X34, a highly fluorescent derivative of Congo red, used for a sensitive detection of pathological amyloid structures (Supplementary Fig. S4). A few labeled neurons were observed in the contralateral neocortex and hippocampus (Fig. 3B–C). Mice treated with AD-derived sarkosyl-insoluble fractions showed double-labeled cells containing AT8-positive deposits and intense P62 labeling (Supplementary Fig. S5), thus suggesting altered proteostasis in AT8-immunoreactive cells after sAD inoculation. No differences in the ptau deposits and their cellular localization were observed when all Prnp genotypes were compared at the analyzed time points.Fig. 3 AT8- and MC1-positive deposits in Prnp+/+; ZH3 Prnp0/0, and GPI− Prnp mice. Immunostaining after sAD inoculation at three months (B–C) and six months (A, E–M) post-inoculation (A) AT8-positive grains (arrows) and threads in the SLM of CA1. The section was Nissl counterstained. (B–C) AT8-positive neurons (arrows in B and C) are seen in the retrosplenial cortex (ipsilateral (B) and contralateral (C)) after sAD inoculation. Sections were Nissl counterstained. (D) Example of AT8-positive grains (arrows) and threads in the SLM of CA1 of ZH3 Prnp0/0. The section was Nissl counterstained. (E–F) MC-1-positive threads and grains (arrows) in the lower cortical layers after sAD inoculation (E) and in the hippocampus of wild-type and ZH3 Prnp0/0 mice. Sections were counterstained with DAPI. (G) Double-immunofluorescence with NeuN (red) and AT8 (green) in lower cortical layers. Note the presence of some ptau deposits in NeuN-positive neurons (arrows). The section was counterstained with DAPI (H–I) Absence of double-labeled GFAP (red) and AT8 (green) cells in the CA1 region of inoculated mice. ZH3 Prnp0/0 (H) and GPI− Prnp (I). (J) AT8 immunofluorescence (green) of axonal tracts in the white matter of the corpus callosum (arrows) of inoculated GPI− Prnp mouse. The section was counterstained with DAPI. (K–M) Double-immunofluorescence showing Olig2-positive oligodendrocytes (red) containing AT8-positive inclusions (green and arrows) in the white matter of ZH3 Prnp0/0 (K), GPI− Prnp (L), and wild-type (M)). Sections were counterstained with DAPI. Scale bars: A–D = 50 µm; E–J = 100 µm; K–M = 50 µm.

A comparison of ptau localization and corpus callosum extension in Prnp+/+, ZH3 Prnp0/0, and GPI− Prnp inoculated mice at the age of 4–5 months and killed three months and six months after inoculation is shown in Fig. 4. At three months after inoculation, AT-8-immunoreactive deposits were seen in the ipsilateral corpus callosum, crossing the midline and extending to the medial part of the contralateral corpus callosum in sections obtained at the level of the site of the injection (Fig. 4A–C). AT8 immunostaining in the corpus callosum was more intense at six months post-inoculation compared to 3 months. Still, the distribution of the AT8-positive deposits in the corpus callosum was the same as at three months (Fig. 4D–F). Similar localization and distribution of AT8-immunoreactive deposits were observed in Prnp+/+, GPI− Prnp0/0, and ZH3 Prnp0/0 mice after injection of AD-derived sarkosyl-insoluble fractions. After quantification of the area occupied by the AT8-positive ptau (Fig. 4G), no significative statistical differences were observed between phenotypes and post-inoculation time although an increased presence was observed between 3- and 6-months post-inoculation as above indicated: (3 months post- inoculation: ZH3 Prnp0/0 = 9606.71 ± 624.17 Prnp+/+  = 10,512.066 ± 482.81; GPI—Prnp0/0 = 8784.19 ± 1171.99. 6 months post-inoculation: ZH3 Prnp0/0 = 13,054.37 ± 1361.18; Prnp+/+  = 14,070.38 ± 1156.85; GPI—Prnp0/0 = 13,394.42 ± 1774.22 (all data is represented as μm2 (mean ± s.e.m.) of AT8-positive deposits/146,850 μm2 photomicrographic field, see Methods for details, and Supplementary Fig. S6 for an example of this analysis in a ZH3 Prnp0/0 photomicrograph).Fig. 4 AT8-positive deposits in the white matter after 3 and 6 months post-sAD inoculation. For each genotype, two schemes are included. In each scheme, the distribution of AT8-positive deposits is illustrated in red dots. A green asterisk indicates the injection site (A); the number of inoculated mice showing the distribution stated in the scheme is displayed in the upper left corner. (A–C) Photomicrographs illustrating the distribution of AT8-positive deposits in the three genotypes three months after sAD inoculation. (D–F) Photomicrographs show AT8-positive deposits in the three genotypes six months after sAD inoculation. The inter-hemispheric fissure is labeled with a dashed line in (A–C). Note the increase in the labeling between 3 and 6 months post-sAD inoculation. The distribution of ptau deposits is similar in the three genotypes and at the two post-inoculation times. (G) Histogram illustrating the results of the quantification of the area occupied by AT8-positive deposits for each of the genotypes and ages (3 or 6 months post-sAD) analyzed. Each point represents the value obtained from one section. See Methods for details and Supplementary Fig. S6 for an example. Values are represented as the mean and s.e.m.. Scale bars: A–C = 250 µm, D = 500 µm; E and F = 500 µm.

AD sarkosyl-insoluble fraction inoculation triggers mouse ptau recruitment and 3Rtau + 4Rtau expression in the host

Inoculation of sAD sarkosyl-insoluble fractions produced seeding and spreading in wild-type mice (Prnp+/+) but not in inoculated Mapt0/0 mice (Fig. 5A,B), thus supporting the endogenous origin of AT8-positive ptau deposits in inoculated animals. The endogenous origin of ptau in inoculated mice was corroborated by immunohistochemistry using Tau13 (human-specific) (Fig. 5C–E, Supplementary Fig. S7A-F) and T49 (mouse-specific) and anti-tau antibodies (Fig. 5F–H, Supplementary Fig. S7G-L). Double-labeling immunostaining showed co-localization of pSer422-positive aggregates and T49 but not pSer422-positive ptau aggregates and Tau13 (Fig. 5C–H, Supplementary Fig. S7A–F).Fig. 5 Characterization of ptau deposits. (A) After sAD inoculation immunostained with the AT8 antibody, a wild-type mouse showed AT8-positive deposits along the corpus callosum. (B) Mapt0/0 mouse three months post-sAD inoculation shows negative AT8 immunostaining. (C–E) Double-labeling immunofluorescence with pSer422 (C) and Tau13 (anti-human tau) (D) showing lack of co-localization. (F–H) Double-labeling immunofluorescence with pSer422 (F) and Tau 49 (anti-mouse tau) (G) shows co-localization of the antibodies, thus indicating the mouse origin of tau in ptau deposits. (I–O) Double-labeling immunofluorescence using pSer422 (I,M) and 3Rtau (J) and 4Rtau (N) specific antibodies show co-localization of pSer422 and 3Rtau, and pSer422 and 4Rtau in deposits. (P–S) High magnifications of the above-mentioned results boxed in (E), (H), (L) and (O). In the Figure, double labelled cells are indicated as arrows and arrowheads point to non-double labelled ptau deposits. The post-inoculation time in C–S was 6 months. The genotype of each inoculated mice is illustrated. Scale bars: A and B = 500µm, C–O = 100µm, P–S = 75μm.

Finally, we aimed to determine whether the observed ptau inclusions contained 3R and 4Rtau isoforms using specific antibodies. Double-labeling immunofluorescence demonstrated the presence of 3R and 4Rtau isoforms in pSer422-positive aggregates (Fig. 5I–S, Supplementary Fig. S8), thus suggesting impaired gene regulation in cells containing abnormal ptau deposits.

Discussion

PrPC involves many functions in the developing and adult brain55–57. PrPC is also involved in neurodegeneration, interacting with several amyloid proteins, thus participating in the pathogenesis of several neurodegenerative diseases with abnormal protein aggregates42–47,49–51,58,59. PrPC has been implicated in the pathological protein aggregates uptake and toxic signaling58. In this line, we showed the involvement of the cellular prion protein in α-synuclein transport in neurons48 in parallel to others studies reviewed in51.

In the present study, after characterization of the sAD homogenates used for inoculation in mice and validation of their suitability as seeding products in Biosensor cells, we have tested the hypothesis that PrPC might play a role in tau seeding and spreading in vivo. As previously reported, we have observed ptau seeding and spreading of inoculated sAD-tau in wild-type mice16,23. In addition to neurons, oligodendrocytes are targets of ptau seeding and ptau propagation60 irrespective of PrPC expression levels and its location (extracellular vs membrane-linked).

Another relevant point has been the observation that 3Rtau and 4Rtau deposits are produced by the host (as revealed with specific anti-mouse and anti-human tau antibodies) following the intracerebral inoculation of sAD (3R + 4Rtau). Similar observations were observed in other paradigms16,23,60,61. Since mice express predominant 4Rtau in the mature brain, the appearance of 3R and 4R tau suggests that exogenous ptau modulates Mapt splicing in the host. A similar shift between 4 and 3Rtau has been described near the ischemic core following middle cerebral occlusion62 Similarly, human tau (4R isoform) packaged into the recombinant AAV serotype 9 inoculated in the rhesus monkey hippocampus, resulted in 3Rtau and 4Rtau accumulation, tau phosphorylation, and propagation two or three months after injection62. However, other studies have not observed a change in the expression of tau isoforms in inoculated mice models24,29,63–65. These findings support the notion that different tau strains produce distinct patterns of neuronal tau deposition and seeding and also that tau seeding depends on the host tau30,60,66,67.

Regarding our main objective, we have used two Prnp mouse transgenic mice, Prnp mice over-expressing the GPI truncated form of prion protein: the “anchorless” GPI− Prnp and ZH3 Prnp0/0, to assess whether PrPC is needed for tau seeding and spreading in vivo. Our results prove that it is not for ptau since both processes occurs in all Prnp genotypes. However, we cannot rule out a minor role of PrPC that could be masked to the relevant function of LRP131,32 or LAG337, as well as other mechanisms in these processes68. In fact, extracellular amyloid proteins mainly prefibrilar or fibrillar treatments can render kinase (e.g., p38, ERK1/2) activation as well other intracellular effects (NADPH activation and ROS generation) in absence of PrPC69–71 that might lead endogenous mouse ptau generation (e.g., see72,73 for reviews). Another scenario, might be related to the fact that although the physical interaction of PrPC mainly with oligomers of Aβ, α-synuclein, tau, and TDP43 has already been described (i.e.,45,48,50,58,59), some studies reported that PrPC cannot bind to oligomeric species of α-synuclein74 or Aβ75, or that their effects in neuronal activity or survival are not linked to PrPC76,77 in contrast to (33,78). In fact, these divergences could be linked to the preparation of the samples since it has been described that fibrils and globular oligomers bind only weakly to PrPC and mediate toxicity in a PrPC-independent manner78 (see also58 for additional comments). However, we believe that our results using sarkosyl-insoluble fractions of AD containing tau seeding properties points a null or minor role of PrPC in seeding and spreading of these sAD fractions in parallel to some of the previously commented studies, in contrast to their relevant role in the progression of inoculated mice with α-synuclein prefibrils48.

Methods

Mouse strains

Adult ZH3 Prnp0/0 mouse line was generated by A. Aguzzi (Switzerland)54. Mice over-expressing the secreted form of PrPC lacking their GPI anchor (GPI− Prnp (Tg44 strain) mice were kindly provided by Vincent Beringue, INRA UR892, Virologie Immunologie Moléculaires, Paris, France)53. Mice lacking tau (Mapt0/079) were kindly provided by Jesús Ávila, CBM-UAM, Madrid). P301S (PS19) transgenic mice80 that over-express the human mutation of tau under the cellular prion protein promoter81 were obtained from Jackson Laboratories ref: 008169. Nestin-cre/Prnpflox/flox mice were generated in our laboratory by crossing Prnpflox/flox82 and Nestin-cre83. A total of 48 adult (2–5 months old) male mice included Prnp+/+ (n = 15), Prnp0/0 (n = 8), GPI− Prnp (n = 11), Mapt0/0 (n = 3), Prnpflox/flox (n = 2) and Nestin-cre/Prnpflox/flox (n = 5). In addition, 4 mice of our P301S colony (12 months of age) were used to obtain sarkosyl-insoluble brain fractions: P301S+/− (n = 2); P301S−/− (n = 2). All the animals were kept in the animal facility at the Faculty of Pharmacy, University of Barcelona, under controlled environmental conditions and were provided food and drink ad libitum. All experiments were performed under the guidelines and protocols of the Ethical Committee for Animal Experimentation (CEEA) of the University of Barcelona; the protocol for the use of animals in this study was reviewed and approved by the CEEA of the University of Barcelona (CEEA approval #276/16 and #141/15), and comply with the guidelines and regulations of ARRIVE essential 10 guidelines 2.084.

Human brain samples

Brain samples were obtained from the Institute of Neuropathology Brain Bank, Bellvitge University Hospital, following the guidelines of the Spanish legislation on this matter (Real Decreto Biobancos 1716/2011) and the approval of the local ethics committee of Bellvitge University Hospital (Hospitalet de Llobregat, Barcelona, Spain). At the time of the autopsy, one hemisphere was fixed in paraformaldehyde. The other hemisphere was cut into coronal Sects. 1 mm thick, and selected brain regions were dissected, immediately frozen at − 80 °C, put on labeled plastic bags, and stored at − 80 °C until use; the rest of the coronal sections were frozen and stored at −80 °C following standard protocols85.

Sarkosyl-insoluble fractions from the frontal cortex (area 8) of ten patients with sAD were characterized. After the clinical, neuropathological, and biochemical study, we selected samples from a 56-year-old female with non-familiar dementia; the genetic study of APP, PSEN1, and PSEN2 revealed no mutations and duplications. The clinical record revealed no infectious diseases, seizures, vascular diseases, and no prolonged agonal state; at post-mortem, the pH of the brain was 6.8. The neuropathological examination categorized sAD as NFT Braak stage VI, Thal phase 4, and CERAD 3; concomitant pathologies including 4Rtaupathies (argyrophilic grain disease: AGD, aging-related tau astrogliopathy: ARTAG), Lewy body disease, limbic-predominant TDP-43 encephalopathy, and hippocampal sclerosis were absent.

In addition, stored sarkosyl-insoluble fractions from AGD, ARTAG and globular glial tauopathy (GGT), healthy non-neurodegenerative cases (already used in previous studies:16,23,27), and extracts from post-mortem brain samples of Parkinson’s disease (PD) and multiple system atrophy (MSA) (Navarra biobank) were employed for comparison in specific experiments.

Sarkosyl-insoluble fraction preparation

Brain tissue was weighed and then homogenized using a Dounce homogenizer in 10 volumes of fresh homogenization buffer (0.8M NaCl, 1mM EGTA, 10% sucrose, 0.01M Na2H2P2O7, 0.1M NaF, 2 mM Na3VO4, 0.025M β-glycerolphosphate, 0.01M Tris–HCl pH 7.4) containing protease inhibitors (Roche, Switzerland). After centrifugation at 16,000 rpm for 22 min at 4 °C, the supernatant was reserved (SN1). The pellet was re-suspended in 5 volumes of homogenization buffer and centrifuged at 14,000 rpm for 22 min at 4 °C. The resulting supernatant (SN2) was then combined with the SN1, and the mixture (SN1 + SN2) was incubated with 0.1% N-lauroyl sarcosinate (sarkosyl; Sigma-Aldrich) and placed on a rotating shaker for one h at room temperature. The mixture was centrifuged at 35,000 rpm for 63 min at 4 °C. The resultant supernatant was discarded, and the remaining pellet (sarkosyl-insoluble fraction) was washed and re-suspended in 50 mM Tris–HCl, pH 7.4 (200 µl/g starting material). Finally, 100 µl aliquots were stored at − 80 °C until use. Protein concentrations were determined using the Pierce™ BCA assay kit (Sigma-Aldrich), and equal amounts of protein were analyzed with western blot.

Biochemical analysis

Following dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting samples were characterized. Protein extracts were boiled at 100 °C for 10 min, followed by SDS-PAGE electrophoresis, and they were then electro-transferred to nitrocellulose membranes for 1 h at 4 °C. Membranes were then blocked with 5% fat milk in 0.1M Tris-buffered saline (pH 7.4) for 1 h and incubated overnight in a 0.5% blocking solution containing the primary antibodies. After incubation with peroxidase-tagged secondary antibodies (at a dilution of 1:2,000, Sigma-Aldrich), the membranes were revealed with an ECL-plus chemiluminescence Western blot kit (Amersham-Pharmacia Biotech).

Tau RD P301S Biosensor cell line experiments

The Tau RD P301S Tau Biosensor (ATCC® CRL-3275™) was purchased from ATCC. Cells were grown in the maintenance medium Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fischer Scientific), supplemented with 10% fetal bovine serum (FBS; Thermo Fischer Scientific), 1% GlutaMax (Gibco), and 1% Penicillin/Streptomycin (Thermo Fischer Scientific) in 75 cm2 culture flasks (Nunc). Cells were maintained at 37 °C and 5% CO2 in a humidified incubator and passaged every three days when confluent. Tau Biosensor cells were plated in 96-well poly-D-lysine (Sigma-Aldrich) (0.1 mg/ml) coated plates at a density of 35,000 cells/well in the maintenance medium (total volume 130 µl) and cultured at 37 °C in a 5% CO2 incubator overnight. The transduction mixture was prepared following the manufacturer’s protocol. Briefly, 1.5 µl of the sample was combined with 8.5 µl of Opti-MEM medium (Thermo Fischer Scientific). A mixture of 1.25 µl Lipofectamine-2000™ reagent (Thermo Fischer Scientific) and 8.75 µl Opti-MEM was added to the sample mixture to a final volume of 20µl and incubated for 1 h at room temperature. Mixtures with empty liposomes were included as negative controls. Tau Biosensor maintenance medium was gently removed and replaced with 130 μl of pre-warmed Opti-MEM before 20 μl the transduction mixture was added to the cells. Twenty-four hours later, cells were washed once with pre-warmed 0.1M PBS and fixed with 4% phosphate-buffered paraformaldehyde (PFA) for 15 min at room temperature. Next, PFA was removed, and cells were washed thrice, 5 min each, with 300 μl of 0.1M PBS. Finally, 300 μl 0.1M PBS with 0.02% NaN3 was placed in each well; the plate was sealed with Parafilm™ and kept at 4 °C until analysis. The following samples were analyzed: fibrillated and monomeric tau K18 (that encompasses the four microtubule-binding repeats (R1-R4) of the tau protein and is one of the minimal sequences necessary for tau aggregation) at a final concentration of 0.01µM (Bio-techno (ref: SP-496-100)); human and murine α-synuclein pre-formed fibrils at 0.1 µg/µl (a gift from Masato Hasegawa, Tokyo Metropolitan Institute of Medical Science, Japan); monomeric tau Cy5 at a final concentration of 100nM (kindly provided by Jesús Ávila, CBM-UAM, Madrid). Sarkosyl-insoluble fractions of P301S+/− and P301S−/− (wild-type) mice were used at a final concentration of 0.003 µg/µl. Sarkosyl-insoluble fractions from human brains were used at a final concentration of 0.003 µg/µl.

Negative electron microscopy staining

For transmission electron microscopy (TEM) experiments, tau samples were fixed to carbon-forward-coated copper grids and negatively stained with buffered 1% uranyl acetate (pH 7.4). The samples were placed in silica-based desiccant for at least two hours and examined with a Jeol JEM-1010 transmission electron microscope.

Mouse tissue homogenization and preparation

Mouse brain tissue of the different strains was weighed and diluted in 1 ml 0.1M PBS supplemented with protease inhibitors (Roche, Switzerland) per 20 mg of tissue. The sample was homogenized for 10 min using a Polytron™. Fifty µl aliquots were stored at − 80 °C until use. Protein concentrations were determined using a BCA assay, and equal amounts of protein were analyzed with western blot.

Stereotaxic surgery

Mice were deeply anesthetized with isoflurane and placed in a stereotaxic apparatus (Kopf Instruments, ref: 963, USA). Unilateral stereotaxic injections were made into the right hippocampus (AP: 1.4 mm from Bregma; LM: 1.5 mm). 2.5 µl of sarkosyl extract dissolved in 100mM Tris-HCl was inoculated using a Hamilton syringe into the upper corpus callosum/cortex at DV of 1 mm86. Following the injection, the needle was kept in place for an additional 3 min. The surgical area was cleaned with sterile saline, and the incision was sutured. Mice were monitored until recovery from anesthesia and were checked regularly following surgery.

Primary antibodies used in the study

The following primary antibodies were used: monoclonal mouse AT8 against pSer202 and pThr205 residues of ptau (1:50 dilution) (Thermo Fischer Scientific, catalog MN1020), polyclonal rabbit anti-tau-phosphoSer422 (pSer422) (1:75 dilution) (Life Technologies, catalog 44-764G), monoclonal mouse anti-PrPC (6H4 clone) (1:1000 dilution) (Thermo Fischer Scientific, catalog 01-010), human tau-specific antibodies Tau13 (1:200 dilution) (Biolegend, catalog no 835201), murine tau-specific antibody T49 (1:200 dilution) (Merck Millipore, catalog no MABN827), and monoclonal mouse MC-1 anti-tau (a gift of Prof. Peter Davis87). Tau isoforms 3R and 4R were assessed using monoclonal mouse antibodies RD3 (clone 8E6/C11) (1:50 dilution) and RD4 (clone 1E1/A6) (1:50 dilution) (Merck Millipore). Other antibodies were polyclonal rabbit anti-P62/SQSTM1 (1:100 dilution) (Progen, ref GP62-C), monoclonal mouse anti-actin (1:1,000 dilution) (Millipore, catalog MAB1501), monoclonal mouse anti-TUJ1 (neuron-specific class III β-tubulin) (1:500 dilution) (BioLegend, catalog 801201).

Tissue processing of sarkosyl-inoculated mice

After 3 (C57BL/6J (n = 10), MAPT0/0 (n = 3), ZH3-Prnp0/0 (n = 5), GPI− Prnp (n = 5)) and 6 (C57BL/6J (n = 5), ZH3-Prnp0/0 (n = 3), GPI− Prnp (n = 6)) months post-injection, mice were processed for neuropathological study. After deep anesthesia with isoflurane, mice were transcardially perfused with phosphate-buffered 4% PFA (pH 7.3) using a peristaltic infusion pump. The brain was dissected and post-fixed overnight in the same fixative. Post-fixed brains were rinsed in 0.1M PBS and stored in 70% ethanol at 4 °C until paraffin inclusion. Following paraffin embedding, 10 µm thick coronal sections were obtained and mounted on gelatinized glass slides. For immunohistochemistry, paraffin sections were de-waxed in xylene for 20 min, washed in ethanol, and rinsed in miliQ H2O. Selected de-waxed sections containing the dorsal hippocampus were treated with DakoTarget retrieval solution (pH 9) (Dako, Denmark) at 95  C in a Dako PT Link to retrieve protein antigenicity. After washing in 0.1M Tris–HCl pH 7.6, sections were rinsed in 0.1M PBS; endogenous peroxidase activity was blocked by incubation in 2% H2O2 and 10% methanol in 0.1M PBS. After rinsing, sections were incubated in 0.1M PBS containing 0.2% gelatin, 10% fetal bovine serum (FBS), 0.2% glycine, and 0.1% Triton X-100 for 1 h at room temperature. Afterward, the sections were incubated with primary antibodies (all primary antibodies were diluted in 5% FBS, 0.1% Triton X-100, and 0.02% NaN3 in 0.1M PBS) overnight at 4 °C. For bright-field visualization, tissue sections were then rinsed in 0.1M PBS and incubated for 2 h at room temperature with species-specific biotinylated secondary antibody (1:200 dilution) (Vector Laboratories) in 0.1M PBS, 5% FBS, 0.1% Triton X-100. Sections were incubated with an avidin–biotin-peroxidase complex (ABC) kit following the manufacturer’s instructions (Vector Laboratories). Peroxidase activity was developed with 0.03% 3–3’-diaminobenzidine (DAB) and 0.01% H2O2. For fluorescence staining, sections were rinsed in 0.1M PBS and incubated with species-specific secondary antibodies, Alexa Fluor-488 or -568 (1:300 dilution) (Life Technologies), for 2 h at room temperature. Then, sections were incubated with 1 µg/ml DAPI (Thermo Fischer Scientific) diluted in 0.1M PBS for 10 min at room temperature, rinsed with 0.1M PBS, and mounted in Mowiol™ (Sigma-Aldrich). Sections were photo-documented using an Olympus BX61 microscope with a cooled digital DP72L camera. As an internal immunohistochemical control for immunohistochemistry, 25 µm thick cryostat sections from the frontal cortex (area 8/9) of AD samples (Braak stage VI) were processed in parallel. For quantification to analyze the degree of ptau seeding after the sAD inoculations, photographs were taken using the 20X objective of areas close to the injection site in the white matter. The images were processed using the ImageJ™ software. For this, the photographed region for each case covered an area of 146,850 μm2 at a resolution of 1360 × 1024 pixels. Under these conditions, each pixel of the image corresponds to an area of 0.1054 μm2. The images processed at 16 bits were analyzed to determine the AT8-positive area using an inverted lut and threshold, and subsequently, the occupied area was calculated using the Particle Analysis plugin of ImageJ™. A minimum of 2–3 images from each case presented in Fig. 4 were processed. The results were processed using the statistical program GraphPad Prism 10.0.1.218 (ID: 97F7835C03E), and a non-parametric ANOVA Dunn’s multiple comparisons analysis was performed.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72232-2.

Acknowledgements

The authors thank Tom Yohannan for his editorial advice. We also thank Miriam Segura-Feliu and Juan José López for their technical help. The authors thank all the members of the Del Río, Ferrer, Ávila, and Aguzzi laboratories for their helpful comments.

Author contributions

J.S.-J., V.G., P.A.-B., I.M.-S., P.P.-P. and J.A.D.R. performed experiments and data analysis. J.A., M.N., J.L.L., A.A., R.G. and I.F. and J.A.D.R. designed the experiments. J.A.D.R. and I.F. write and edited the manuscript. All authors contributed to the article and approved the submitted version.

Funding

J.A. del Río and R. Gavín were supported by PRPCDEVTAU PID2021-123714OB-I00, ALTERNed PLEC2022-009401, PDC2022-133268-I00 and ADNano from Plan Complementario de Biotecnología Aplicada a la Salud (C17.I1) funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”, the CERCA Programme, and by the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (SGR2021-00453). The project leading to these results received funding from “la Caixa” Foundation (ID 100010434) under the agreement LCF/PR/HR19/52160007 to JADR and IF. V. Gil was supported by the María de Maeztu Unit of Excellence (Institute of Neurosciences, University of Barcelona) MDM-2017-0729. J. Sala-Jarque was supported by the Tatiana Pérez de Guzmán el Bueno Foundation. I. Martínez-Soria was supported by MICINN (FPI Programme, PRE2022-103197) and P. Picón-Pagès was supported by Ciberned.

Data availability

The data supporting the conclusions of this article will be made available by the corresponding author upon request, without undue reservation.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Julia Sala-Jarque and Vanessa Gil.

These authors jointly supervised this work: Rosalina Gavín, Isidro Ferrer and José Antonio del Río.
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