Dysregulated protein phosphorylation in tauopathy

doi:10.21203/rs.3.rs-1442779/v1

The neocortex of P301S mice, used as a model of frontotemporal degeneration linked to tau mutation, and wild-type mice, both aged 9 months, were analyzed with conventional label-free phosphoproteomics and SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) to assess the (phospho)proteomes. The total number of identified dysregulated phosphoproteins was 328 corresponding to 524 phosphorylation sites. The majority of dysregulated phosphoproteins, most of them hyper-phosphorylated, were proteins of the membranes, synapses, membrane trafficking, membrane vesicles linked to endo- and exocytosis, cytoplasmic vesicles, and cytoskeleton. Another group was composed of kinases. In contrast, proteins linked to DNA, RNA metabolism, RNA splicing, and protein synthesis were hypo-phosphorylated. Other pathways modulating energy metabolism, cell signalling, Golgi apparatus, carbohydrates, and lipids are also targets of dysregulated protein phosphorylation in P301S mice.

The present results, together with accompanying immunohistochemical and western blotting studies, show widespread abnormal phosphorylation of proteins, in addition to protein tau, in P301S mice. These observations point to dysregulated protein phosphorylation as a relevant contributory pathogenic component of tauopathies.

tau

(phospho)proteomics

tauopathy

membranes

cytoskeleton

kinases

Neurodegenerative diseases with abnormal protein aggregates are characterized neuropathologically by the deposition of protein aggregates resulting mainly from aberrant post-translational modifications of primary constitutive elements of the nervous system. Alzheimer’s disease (AD), characterized by the accumulation of β-amyloid and abnormal tau species, is a paradigm of this group of disorders. Senile plaques, β-amyloid angiopathy, neurofibrillary tangles (NFTs), dystrophic neurites, and neuropil threads are the morphological hallmarks of β-amyloid and tau deposits in AD [1–5]. Primary tauopathies are heterogeneous diseases having in common the deposition of abnormally phosphorylated species of tau protein, usually accompanied by other post-translational modifications in tau in neurons and glial cells; various types of neuronal, astroglial and oligodendroglial tau inclusions define different tauopathies. Tauopathies are sporadic, or familial linked to mutations in MAPT, the tau gene [6–11].

Phosphorylation is one of the most common and essential mechanisms of protein function that mostly derives from activation/inhibition of protein function and/or recruitment of interacting proteins with structurally conserved domains [12–17].

AD generates, in addition to hyperphosphorylated tau, dysregulated phosphorylation of a large number of proteins [18–20]. Differentially regulated phosphoproteins are components of cell membranes and membrane signalling, cytoskeleton, synapses including neurotransmitter receptors, serine-threonine kinases, proteins involved in energy metabolism, and RNA processing and splicing, among others [18, 19, 21]. Dysregulated protein phosphorylation in AD occurs at the first stages of NFT pathology, and precedes, in the frontal cortex, the appearance of abnormal β-amyloid and tau deposits in this region [21]. Abnormal phosphorylation of various proteins occurs in aging-related tau astrogliopathy (ARTAG) [22]. Large-scale (phospho)proteomics also reveals dysregulated protein phosphorylation in globular glial tauopathy (GGT), a rare sporadic and familial tauopathy [23]. Dysregulated protein phosphorylation has also been reported in transgenic mouse models of AD expressing abnormal β-amyloid deposition [24–29], but there is little information about differential protein phosphorylation in transgenic models of pure tauopathy [26]. For this reason, the present study is focused on altered protein phosphorylation in a validated model of primary tauopathy linked to P301S mutation in the tau gene. Our hypothesis is that in addition to abnormal tau phosphorylation, dysregulated protein phosphorylation involving key proteins is a common and relevant molecular pathology in tauopathies.

Animals

The experiments were carried out in heterozygous transgenic mice expressing human P301S tau (line PS19: B6;C3-Tg(Prnp-MAPT*P301S)PS19Vle/J), and wild-type (WT) littermates in a C57BL/6 background. The number of mice was nine per group with equal numbers of males and females per group. Transgenic mice were identified by genotyping genomic DNA isolated from tail clips using the polymerase chain reaction conditions indicated by Jackson Laboratory (Bar Harbor, ME). Animals were maintained under standard animal housing conditions in a 12-hour dark-light cycle with free access to food and water. All animal procedures were carried out following the guidelines of the European Communities Council Directive 2010/63/EU and with the approval of the local ethical committee (C.E.E.A: Comitè Ètic d’Experimentació Animal; University of Barcelona, Spain; ref. 426/18). Animals were killed at the age of nine months by cervical dislocation and their brains were then rapidly removed and processed for study. The left cerebral hemisphere was dissected on ice, immediately frozen, and stored at -80°C until use for biochemical studies. The right hemisphere, brainstem, and cerebellum were fixed in 4% paraformaldehyde, cut in coronal sections, and embedded in paraffin. De-waxed sections were stained with haematoxylin and eosin, or processed for immunohistochemistry.

Immunohistochemistry

De-waxed sections, 4 microns thick, were processed for immunohistochemistry. The sections were boiled in citrate buffer pH = 6 (20 min) to retrieve non-specific antigenicity. Endogenous peroxidases were blocked by incubation in 10% methanol-1% H₂O₂ solution (15 min), followed by 3% normal horse serum solution. The sections were incubated at 4ºC overnight with one of the primary antibodies listed in Table I. The antibodies were the same as those used for the study of hTau transgenic mice, and tau seeding and spreading following inoculation of sAD tau in the hippocampus in those mice [30]. After incubation with the primary antibody, the sections were incubated with EnVision + system peroxidase (Dako, DK) for 30 min at room temperature. The peroxidase reaction was visualized with diaminobenzidine and H₂O₂. Control of immunostaining included omission of the primary antibody; no signal was obtained following incubation with only the secondary antibody.

Double-labeling Immunofluorescence And Confocal Microscopy

De-waxed sections, 4 microns thick, were stained with a saturated solution of Sudan black B (Merck) for 15 min to block autofluorescence of lipofuscin granules present in cell bodies, and then rinsed in 70% ethanol and washed in distilled water. The sections were boiled in citrate buffer to enhance antigenicity and blocked for 30 min at room temperature with 10% fetal bovine serum diluted in PBS. Then, the sections were incubated at 4ºC overnight with combinations of primary antibodies against different proteins. The characteristics of the antibodies, the dilutions, and the suppliers are listed in Table 1. After washing, the sections were incubated with Alexa488 or Alexa546 (1:400, Molecular Probes) fluorescence secondary antibodies against the corresponding host species. Nuclei were stained with DRAQ5™ (1:2,000, Biostatus). After washing, the sections were mounted in Immuno-Fluore mounting medium (ICN Biomedicals), sealed, and dried overnight. Sections were examined with a Leica TCS-SL confocal microscope.

Table 1

List of antibodies used for immunohistochemistry and western blotting: 3Rtau: tau with three repeats; 4Rtau: tau with four repeats; AKT1: serine/threonine-protein kinase AKT1; β-actin; CK1-δ: casein kinase δ; eIF2α: eukaryotic initiation factor 2α; GSK β: glycogen synthase kinase 3β; IRE1: inositol-requiring enzyme 1α; LAMP1: lysosomal-associated membrane protein 3; LC3: microtubule-associated protein 1A/1B-light chain 3; MAP2: microtubule-associated protein 2; NFL: neurofilament light molecular weight; p38: p38-kinase; PHF1: anti-paired helical filament 1; PKAα/β: cAMP-dependent protein kinase A; SAPK/JNK: stress-activated protein kinase/Jun amino-terminal kinase; SRC: tyrosine-protein kinase SRC; Tau 5: monoclonal recognizing total tau protein; Tau AT8; Tau C3: tau truncated at aspartic acid 421; Tau MC1: conformation tau; Tau-N: nitrated tau; Tau-P phosphorylated tau. The phosphorylation or nitration of the protein at the specific site is indicated in every case. WB dil: western blotting dilution; IHQ dil: immunohistochemistry dilution.
Antibody	Supplier	Reference	Host		IHQ dil
3Rtau	Upstate	05-803	Ms	1/1,000	1/800
4Rtau	Millipore	05-804	Ms	1/1,000	1/50
AKT1-P Ser473	Abcam	Ab18206	Rb	-	1:100
β-actin	Sigma	A5316	Ms	1/30,000	-
CK1-δ	Abcam	ab85320	Ms	-	1/500
eIF2α-P Ser51	Thermo Scientific	MA5-15133	Rb	-	1/100
GFAP	Dako	Z0334	Rb	-	1/400
GSK-3β-P Ser9	Cell signaling	9336	Rb	-	1/100
Iba1	Wako	019-19741	Rb	-	1/1000
IRE-P Ser274	Abcam	ab48187	Rb	-	1/100
LAMP1	Santa Cruz	Sc5570	Rb	-	1/10
LC3	Cell Signalling	2775	Rb	-	1/100
MAP2-P Thr1620/1623	Cell Signaling	4544	Rb	-	1/1,000
NFL-P Ser473	Millipore	MABN2431	Ms	-	1/100
p38-P Thr180/Tyr182	Cell Signaling	9211	Rb	-	1/100
PHF1, tau-P Ser396/Ser404	Dr. Peter Davies	-	Ms	-	1/500
PKAα/β-P Tyr197	Invitrogen	44988	Rb		1/100
SAPK/JNK-P-Thr183/Thr185	Cell Signaling	9251	Rb	-	1/25
SRC-P Tyr416	Millipore	05-677	Ms	-	1/100
SRPK1	Abcam	ab90527	Rb	-	1/100
SRPK2-P Ser497	Affbiotech	3632	Rb	-	1/100
Tau 5	Thermo Scientific	MA5-12808	Ms	1/500	-
Tau AT8-P Ser202/Thr205	Innogenetics	90206	Ms	-	1/50
Tau C-3	Millipore	36 − 017	Ms	-	1/100
Tau MC1	Dr. Peter Davies		Ms	1/100	1/20
Tau-N Tyr29	Millipore	MAB2244	Ms	-	1/200
Tau-P Ser422	Thermo Scientific	44764	Rb	-	1/50
Tau-P Thr181	Thermo Scientific	PA1-14413	Rb	1/1,000	-
Ubiquitin	Dako	Z0458	Ms	-	1/250

Gel Electrophoresis And Western Blotting

Frozen samples of the posterior part of the left hemisphere from 3 WT and 4 P301S mice were homogenized in RIPA lysis buffer composed of 50mM Tris/HCl buffer, pH 7.4 containing 2mM EDTA, 0.2% Nonidet P-40, 1mM PMSF, protease, and phosphatase inhibitor cocktail (Roche Molecular Systems, USA). The homogenates were centrifuged for 20 min at 12,000 rpm. Protein concentration was determined with the BCA method (Thermo Scientific). Equal amounts of protein (12µg) for each sample were loaded and separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose membranes (Amersham, Freiburg, GE). Non-specific bindings were blocked by incubation in 3% albumin in PBS containing 0.2% Tween for 1 h at room temperature. After washing, membranes were incubated overnight at 4°C with antibodies against different forms of tau protein (Table I). Protein loading was monitored using an antibody against β-actin (42kDa, 1:30,000, Sigma). Membranes were incubated for 1 h with appropriate HRP-conjugated secondary antibodies (1:3,000, Dako); the immunoreaction was revealed with a chemiluminescence reagent (ECL, Amersham). Results were analyzed statistically with SPSS 19.0 (SPSS Inc., USA) software and GraphPad PRISM (GraphPad Software, Inc.) software. Data were presented as mean ± standard error of the mean (SEM). The unpaired student’s t-test was used to compare groups. Significance level was set at * P < 0.05 ** P < 0.01, *** P < 0.001.

(Phospho)proteomic Analysis

Brain samples of the anterior left hemisphere from WT and P301S mice (n = 8) were homogenized in a lysis buffer containing 7M urea, 2M thiourea, 50mM DTT supplemented with protease, and phosphatase inhibitors. The homogenates were spun down at 100,000 x g for 1 hour at 15°C. Protein quantitation was performed with the Bradford assay kit (Bio-Rad). The (phospho)proteomes and the corresponding proteomes were independently analyzed by conventional label-free phosphoproteomics [31] and SWATH-MS (Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) [32], respectively.

Label-free phosphoproteomics: 600µg of protein was used to obtain the phosphorylated fractions. For protein digestion, reduction was performed by addition of DTT to a final concentration of 10mM and incubation at RT for 30 minutes. Subsequent alkylation with 30mM (final concentration) iodoacetamide was performed for 30 minutes in the dark at room temperature. An additional reduction step was performed with 30mM DTT (final concentration), allowing the reaction to stand at room temperature for 30 minutes. The mixture was diluted to 0.6M urea using MilliQ-water, and after addition of trypsin (Promega) (enzyme:protein, 1:50 w/w), the sample was incubated at 37ºC for 16h. Digestion was quenched by acidification (pH < 6) with acetic acid. After protein enzymatic cleavage, peptide cleaning was performed using Pierce™ Peptide Desalting Spin Columns (ThermoFisher). To obtain the phosphorylated peptide fractions, the High-Select™ TiO₂ Phosphopeptide Enrichment Kit (Thermo Scientific) was used according to the manufacturer’s instructions. Phosphopeptide mixtures were separated by reversed-phase chromatography using an Eksigent NanoLC ultra 2D pump fitted with an Acclaim™ PepMap™ 100 C18 column (0.075x250 mm, particle size 3µm; ThermoFisher). Samples were first loaded for concentration into an Acclaim™ PepMap™ 100 C18 trap column (0.1x20 mm, particle size 5µm; ThermoFisher). Mobile phases were 100% water 0.1% formic acid (FA) (buffer A), and 100% Acetonitrile 0.1% FA (buffer B). Column gradient was developed in a gradient from 2% B to 40% B in 120 min. Column was equilibrated in 95% B for 10 min and 2% B for 10 min. During the entire process, the precolumn was in line with the column and flow maintained all along the gradient at 300nl/min. Eluting peptides were analyzed using a 5600 Triple-TOF mass-spectrometer (Sciex). Information data acquisition was acquired upon a survey scan performed in a mass range from 350 m/z up to 1250 m/z in a scan time of 25ms. Top 15 peaks were selected for fragmentation. Minimum accumulation time for MS/MS was set to 200 ms giving a total cycle time of 3.3 s. Product ions were scanned in a mass range from 100 m/z up to 1500 m/z and excluded for further fragmentation during 15 s. The raw MS/MS spectra searches were processed using the MaxQuant software (v 1.6.7.0) and searched against the Uniprot proteome reference for Mus Musculus (Proteome ID: UP000000_10090, March 2021). The parameters used were as follows: initial maximum precursor (15 ppm) fragment mass deviations (20 ppm); fixed modification (Carbamidomethyl (C)); variable modification (Oxidation (M); Acetyl (Protein N-terminal; Phospho (STY)); enzyme (trypsin) with a maximum of 2 missed cleavage; minimum peptide length (7 aminoacids); and false discovery rate (FDR) for PSM and protein identification (1%). Frequently observed laboratory contaminants were removed. Perseus software (version 1.6.14.0) was used for statistical analysis and data visualization.

SWATH-MS: For protein expression analysis, a pool containing the same amount of µg per sample was used as input for the generation of the SWATH-MS assay library. Twenty micrograms were diluted in Laemmli buffer and loaded into a 0.75 mm thick polyacrylamide gel with a 4% stacking gel cast over a 12.5% resolving gel. Total gel was stained with Coomassie Brilliant Blue, and 12 equal slides from each pooled sample were excised from the gel and transferred into 1.5mL Eppendorf LoBind tubes. Protein enzymatic cleavage was carried out with trypsin (Promega; 1:20, w/w) at 37°C for 16 h as previously described [33]. Purification and concentration of peptides were performed using C18 Zip Tip Solid Phase Extraction (Millipore). The peptides recovered from in-gel and in-solution digestion processing were reconstituted into a final concentration of 0.5µg/µL of 2% ACN, 0.5% FA, 97.5% MilliQ-water prior to mass spectrometric analysis. MS/MS datasets for spectral library generation were acquired on a TripleTOF 5600 + mass spectrometer (Sciex) interfaced to the Eksigent nanoLC ultra 2D pump system (Sciex) as previously described. MS/MS data acquisition was performed using AnalystTF 1.7 (Sciex), and spectra files were processed through ProteinPilot v5.0 search engine (Sciex) using the Paragon™ Algorithm (v.4.0.0.0) [34] for database search. To avoid using the same spectral evidence in more than one protein, the identified proteins were grouped based on MS/MS spectra with the Progroup™ Algorithm, regardless of the peptide sequence assigned. False discovery rate (FDR) was determined using a non-lineal fitting method [35] and displayed results were those reporting a 1% global FDR or better. Then, individual protein extracts from all sample sets (n = 8) were subjected to in-solution digestion, peptide purification, and reconstitution prior to mass spectrometric analysis. Protein extracts (20g) from each sample were reduced, adding DTT to a final concentration of 10mM and incubation at room temperature. Reduction was performed by the addition of DTT to a final concentration of 10mM and incubation at room temperature for 30 minutes. Subsequent alkylation by 30 mM iodoacetamide was performed for 30 minutes in the dark. An additional reduction step was performed with 30mM DTT, allowing the reaction to stand at room temperature for another 30 minutes. The mixture was then diluted to 0.6M urea using MilliQ-water, and after addition of trypsin (Promega) (enzyme:protein, 1:50 w/w), the sample was incubated at 37ºC for 16h. Digestion was quenched by acidification with acetic acid. The digestion mixture was dried in a SpeedVac. Purification and concentration of peptides was made using C18 Zip Tip Solid Phase Extraction (Millipore). The peptides recovered were reconstituted into a final concentration of 1µg/µL of 2% ACN, 0.5% FA, 97.5% MilliQ-water prior to mass spectrometric analysis. For SWATH-MS-based experiments, the TripleTOF 5600 + instrument was configured as described [36]. Using an isolation width of 16Da (15Da of optimal ion transmission efficiency and 1Da for the window overlap), a set of 37 overlapping windows was constructed covering the mass range 450–1000 Da. In this way, 1µL of each sample was loaded into an Acclaim™ PepMap™ 100 C18 trap column (0.1x20 mm, particle size 5 µm; ThermoFisher) and desalted with 100% water 0.1% formic acid at 2µL/min for 10 min. The peptides were loaded into an Acclaim™ PepMap™ 100 C18 column (0.075x250 mm, particle size 3µm; ThermoFisher) equilibrated in 2% acetonitrile 0.1% FA. Peptide elution was carried out with a linear gradient of 2 to 40% B in 120 min (mobile phases A:100% water 0.1% formic acid (FA) and B: 100% acetonitrile 0.1% FA) at a flow rate of 300nL/min. Eluted peptides were infused in the mass-spectrometer. The Triple TOF was operated in SWATH mode, in which a 0.050s TOF MS scan from 350 to 1250m/z was performed, followed by 0.080s product ion scans from 230 to 1800 m/z on the 37 defined windows (3.05s/cycle). Collision energy was set to optimum energy for a 2 + ion at the center of each SWATH block with a 15 eV collision energy spread. The resulting ProteinPilot group file from library generation was loaded into PeakView® (v2.1, Sciex), and peaks from SWATH runs were extracted with a peptide confidence threshold of 99% confidence (Unused Score ≥ 1.3) and FDR lower than 1%. For this, the MS/MS spectra of the assigned peptides were extracted by ProteinPilot, and only the proteins that fulfilled the following criteria were validated: (1) peptide mass tolerance lower than 10ppm, (2) 99% confidence level in peptide identification, and (3) complete b/y ions series found in the MS/MS spectrum. Only proteins quantified with at least two unique peptides were considered. Then, quantitative data were analysed using the Perseus software (version 1.6.14.0) for statistical analysis and data visualization. MS data and search result files were deposited in the Proteome Xchange Consortium via the JPOST partner repository (https://repository.jpostdb.org, accessed on 1 March 2022) [37], with the identifier PXD031961 for ProteomeXchange and JPST001509 for jPOST (https://repository.jpostdb.org/preview/1738288420621e76ca4fcef; Access key 8489)

Bioinformatics: The identification of significantly dysregulated regulatory/metabolic pathways in proteomic datasets was performed using Metascape [38]. Network analysis was performed submitting the corresponding protein IDs to the STRING (Search Tool for the Retrieval of Interacting Genes) software (http://stringdb.org/) [39]. Proteins are represented with nodes and all the edges were supported by at least one reference from the literature or from canonical information stored in the STRING database. To minimize false positives as well as false negatives, only interactions tagged as “medium confidence” (> 0.4) in STRING database were considered.

P301S transgenic mice

Transgenic mice at the age of 9 months showed phosphorylated tau deposition, as revealed with the AT8 antibody, in the cytoplasm of neurons and proximal dendrites in the entorhinal and piriform cortex, CA1 region of the hippocampus, dentate gyrus, somatosensory cortex, and cingulate cortex; the amygdala, hypothalamus, striatum, thalamus, septal nuclei, nuclei of the basal forebrain, and granule cells of the cerebellum were also immunostained with AT8 antibodies; neurons of the brainstem, particularly those in the pontine nuclei, tegmentum, and raphe nuclei also contained AT8-positive deposits (Fig. 1A-C). Neuropil threads in the grey matter, plexiform layers of the hippocampus, hilus of the dentate gyrus, and molecular layer of the cerebellum also contained AT8-positive deposits. Antibodies PHF1, directed against tau-P Ser396/404, and phospho-tau Ser422 showed a similar distribution and localization of phosphorylated tau (Fig. 1D-H). However, the antibody MC1, directed against conformational tau isoforms, stained lesser numbers of neurons in all regions (Fig. 1I); and the antibody tau C-3, directed against truncated tau at aspartic acid 421, stained only a few neurons in the CA1 region and dentate gyrus (Fig. 1J). Tau deposits were homogeneous and diffuse and did not have the appearance of neurofibrillary tangles. Astrocytes and oligodendrocytes were devoid of phosphorylated tau deposits. These findings were in agreement with our previous observations in P301S transgenic mice [40].

Immunohistochemistry with anti-4Rtau antibodies revealed a positive neuropil, but not increased 4Rtau deposits in any cell type. 3Rtau immunohistochemistry was negative. Neurons also contained deposits of tau nitrated at Tyr29 (tau-N Tyr29) in the same regions and localizations like those revealed with AT8 antibodies (Fig. 1K, L). Neurons also contained phosphorylated microtubule associated protein, as recognized with the MAP2-P Thr1620-1623 antibody, with the same extension and localization as tau AT8 (Fig. 2A-C). In contrast, only very rarely did neurons localized in the hippocampus and cerebral cortex exhibit phosphorylated light neurofilament (NFL-P Ser473) immunoreactivity (Fig. 2D). A subpopulation of neurons was positive with anti-casein kinase δ antibodies (Fig. 2E, F). Subpopulations of neurons also showed cytoplasmic granules positive with antibodies against active p38 (p38-P Thr180/tyr182), phosphorylated tyrosine-protein kinase SRC (SRC-P Tyr416) (only in the entorhinal cortex), and phosphorylated stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK-P Thr183/185) (Fig. 2G-J). GSK-3β-P Ser9 immunoreactivity was negative. Markers of reticulum stress eIF2α-P Ser51 and IRE-P Ser274 were negative. Only a few neurons in the dentate gyrus and threads in the hilus showed ubiquitin deposits (Fig. 2K). LC3-immunoreactive granules were not observed. Immunostaining with AKT-P Ser473, PKAα/β-P Tyr197, SRPK1, and SRPK2-P Ser497 antibodies was negative. SRPK1 immunoreactivity was localized in neuronal and glial cell nuclei; inclusions were negative.

Double-labelling immunofluorescence and confocal microscopy to AT8 and active p38 showed co-localization of tau deposits and the active tau kinase in the cytoplasm (Fig. 3A, B). Similarly, neurons with AT8-immunoreactive deposits co-localized SAPK/JNK-P-positive granules in the cytoplasm (Fig. 3C, D).

WT mice showed weak 4Rtau immunoreactivity in the neuropil. The AT8 antibody revealed only nuclear immunostaining without positivity in the cytoplasm or dendrites of neurons. Immunohistochemistry with the rest of the antibodies did not show abnormal deposits.

GFAP-immunoreactive astrocytes were abundant in the CA1 region and dentate gyrus when compared with age-matched WT controls. Microglial cells, as revealed with the Iba1 antibody, showed a significant increase in the CA1 subfield of the hippocampus and dentate gyrus in P301S mice. Glial responses were in line with our previous observations in these mice [40].

Western Blotting Of Total Brain Homogenates In Wt And P301s

Western blots of the anterior part of the left cerebral hemisphere were carried out in 3 WT and 4 P301S transgenic mice. The antibody Tau 5, which identifies total tau, showed strong bands of variable molecular weight in P301S when compared with WT mice. 4Rtau was present in WT mice, but it was strongly increased in P301S, showing two bands of 68kDa and 64kDa. In contrast, 3Rtau was absent in WT and P301S mice. Phosphorylated tau at Thr181 and MC1 tau were expressed in P301S, but they were almost absent in WT mice. Curiously, a doublet of about 64kDa was observed with the antibodies P-tau 181 and MC1 in three mice, but a unique upper band of 68kDa was found in the fourth P301S mouse (Fig. 4).

(Phospho)proteomics

The increment in the tau phosphorylation of four serines (positions 396, 404, 422 and 202) previously observed by immunohistochemistry, were confirmed by mass-spectrometry (Supplementary Table 1). Four additional residues (S400, S416, S198, S199) were also hyperphosphorylated in P301S transgenic mice. Moreover, 524 phosphosites were also identified by our phosphoproteomic approach, with 215 being hypo-phosphorylated and 309 hyper-phosphorylated (Fig. 5A). Three-hundred twenty-eight phosphoproteins were differentially regulated in P301S transgenic mice when compared with WT mice; 179 up-regulated, 134 down-regulated, and 16 both up-regulated and down-regulated in different phosphorylation sites. Fifty-five percent of the differentially regulated phosphoproteins were quantified at the level of the total protein, showing that the levels of phosphorylation were not dependent on the total protein levels. Regarding the remaining 45%, total levels of these proteins were not quantified, and we cannot assume that 100% of the differences were due exclusively to phosphorylation. Enrichment analysis of obtained data revealed that the main differentially regulated phosphoproteins were categorized with the cellular component GO terms related to (i) synapse, including presynapse (GO:0098793) and postsynapse (GO:0097481), and related components (Fig. 5B) such as exocytic vesicle (GO:0070382), glutamatergic synapse (GO:0098978) or synapse vesicle (GO:0099003); (ii) structural components of synaptic and neuronal architecture and cytoskeleton (Fig. 5B), such as axon (GO:0030424), dendrite (GO:0030425), neuronal cell body (GO:0043025), cell junction (GO:0030054), and neuron projection (GO:0043005); and also (iii) membrane and vesicle terms (Fig. 5B), such as cytoplasmic vesicles (GO:0016023); endomembrane system (GO:0012505) or plasma membrane region (GO:0098590) (Fig. 5C).

Identification Of Differentially Regulated Phosphoproteins

The pinpoints of the differentially regulated proteins were assessed manually using available databases.

Two-hundred eighty nine differentially regulated phosphoproteins were classified according to their localization and function into six major groups: kinases; proteins linked to the cytoskeleton; proteins linked to the cellular membranes, membrane metabolism, synapsis, vesicles, endocytosis, and exocytosis; proteins linked to the DNA, RNA, and protein synthesis; proteins linked to autophagy and ubiquitin-proteasome system (UPS); and phosphatases. Other differentially regulated phosphoproteins were involved in cell signalling, energy metabolism, and lipid and carbohydrate metabolism, among other functions.

The remaining 39 differentially regulated phosphoproteins were not considered in the study because their function in the nervous system was barely known.

Thirty-seven differentially regulated phosphoproteins were kinases: 23 were up-regulated, 12 down-regulated, and two had both up-regulated and down-regulated phosphorylation sites in P301S compared with WT mice (Table 2).

Table 2

List of altered phosphoproteins in P301S in comparison with WT mice related to kinases and their functions. The number of dysregulated phosphosites and direction (hyper- or hypophosphorylation) are indicated for each protein.
Protein name		Function	Phosphosite deregulation
Aak1	AP2-associated protein kinase 1 (EC 2.7.11.1) (Adaptor-associated kinase 1)	Membrane signalling	1↑1↓
Akap12	A-kinase anchor protein 12 (AKAP-12) (Src-suppressed C kinase substrate) (SSeCKS)	Compartmentation of PKA and PKC	1↑
Braf	Serine/threonine-protein kinase B-raf (Fragment)	Cytoskeleton	1↑
Brsk1	Serine/threonine-protein kinase BRSK1	Cytoskeleton	3↑
Brsk2	Serine/threonine-protein kinase BRSK2	Cytoskeleton	4↑
Camk2a	Calcium/calmodulin-dependent protein kinase type II subunit alpha (CaM kinase II subunit alpha) (EC 2.7.11.17)	Synaptic plasticity, neurotransmitter release	3↓
Camk2b	Calcium/calmodulin-dependent protein kinase (EC 2.7.11.17)	Actin interaction	4↓
Camk2g	Calcium/calmodulin-dependent protein kinase type II subunit gamma (Fragment)	Actin interaction	2↑
Cdk16	Cyclin-dependent kinase 16 (EC 2.7.11.22)	Vesicle/excocytosis	1↑
Cdkl5	Cyclin-dependent kinase-like 5	BDNF pathway	1↑
Git1	ARF GTPase-activating protein GIT1 (ARF GAP GIT1) (G protein-coupled receptor kinase-interactor 1)	Spine morphogenesis	3↑1↓
Gsk3a	Glycogen synthase kinase-3 alpha (GSK-3 alpha) (EC 2.7.11.26)	Glycogen metabolism, tau phosphorylation	1↑
Limk1	LIM domain kinase 1 (Fragment)	Cytoskeleton	2↑
Lmtk2	Serine/threonine-protein kinase LMTK2 (EC 2.7.11.1)	Endosomal trafficking	1↓
Map3k7	Mitogen-activated protein kinase kinase kinase 7 (EC 2.7.11.25)	Cytoskeleton	1↑
Mapk1	Mitogen-activated protein kinase 1 (MAP kinase 1) (MAPK 1) (EC 2.7.11.24)	Cytoskeleton	1↑
Mapk10	Mitogen-activated protein kinase (EC 2.7.11.24)	Cytoskeleton	1↑
Marcks	Myristoylated alanine-rich C-kinase substrate (MARCKS)	Cytoskeleton	3↑
Mark1	Serine/threonine-protein kinase MARK1 (EC 2.7.11.1) (EC 2.7.11.26)	Cytoskeleton	1↑
Mink1	Misshapen-like kinase 1 (EC 2.7.11.1)	Cytoskeleton	1↑
Nucks1	Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (JC7)	Nuclear	1↓
Nyap2	Neuronal tyrosine-phosphorylated phosphoinositide-3-kinase adapter 2	Morphogenesis	2↑
Pak1	Non-specific serine/threonine protein kinase (EC 2.7.11.1)	Membrane/synapsis/GABA receptor	2↑
Phka2	Phosphorylase b kinase regulatory subunit	Glycogen metabolism	1↑
Pi4kb	Phosphatidylinositol 4-kinase beta	Golgi complex	1↑
Pikfyve	1-phosphatidylinositol 3-phosphate 5-kinase (EC 2.7.1.150)	Golgi/transport	1↑
Prkab1	5'-AMP-activated protein kinase subunit beta-1 (AMPK subunit beta-1) (AMPKb)	Energy metabolism	1↓
Prkacb	cAMP-dependent protein kinase catalytic subunit beta (PKA C-beta) (EC 2.7.11.11)	DNA/RNA	1↓
Prkar1a	cAMP-dependent protein kinase type I-alpha regulatory subunit [Cleaved into: cAMP-dependent protein kinase type I-alpha regulatory subunit, N-terminally processed]	Glucose/lipid metabolism	1↓
Prkce	Protein kinase C epsilon type (EC 2.7.11.13) (nPKC-epsilon)	Membrane signalling	2↓
Prkcg	Protein kinase C gamma type (PKC-gamma) (EC 2.7.11.13)	Membrane signalling	1↓
Prpf4b	Serine/threonine-protein kinase PRP4 homolog (EC 2.7.11.1)	DNA/RNA	3↓
Rps6kc1	Non-specific serine/threonine protein kinase (EC 2.7.11.1)	Endosomes	1↑
Spag9	C-Jun-amino-terminal kinase-interacting protein 4 (JIP-4) (JNK-interacting protein 4)	Cell signalling/c-Jun kinase activity	1↓
Sphkap	A-kinase anchor protein SPHKAP (SPHK1-interactor and AKAP domain-containing protein)	PKA-binding protein	1↑
Srcin1	SRC kinase-signaling inhibitor 1	Synapsis/cell signalling	3↑
Stk32c	Serine/threonine-protein kinase 32C (EC 2.7.11.1)	Cell signalling	1↓

Thirty-four differentially regulated phosphoproteins were related to the cytoskeleton, 18 up-regulated, 12 down-regulated, and four with both up- and down-regulated phosphorylation sites in P301S compared with WT mice (Table 3).

Table 3

List of altered phosphoproteins in P301S in comparison with WT mice related to the cytoskeleton in addition to tau protein. The number of dysregulated phosphosites and direction (hyper- or hypophosphorylation) are indicated for each protein.
Protein name		Phosphosite deregulation
Ablim2	Actin-binding LIM protein 2	3↓
Add1	Alpha-adducin (Erythrocyte adducin subunit alpha)	3↓1↑
Anln	Anillin	2↓
Apc	Adenomatous polyposis coli protein	1↑
Atat1	Alpha-tubulin N-acetyltransferase 1 (Alpha-TAT) (Alpha-TAT1) (TAT) (EC 2.3.1.108) (Acetyltransferase mec-17 homolog)	1↑
Camsap2	Calmodulin-regulated spectrin-associated protein 2	1↑
Cap2	Adenylyl cyclase-associated protein 2 (CAP 2)	1↓
Capza2	F-actin-capping protein subunit alpha-2 (CapZ alpha-2)	1↓
Caskin1	Caskin-1 (CASK-interacting protein 1)	2↑
Cep170	Centrosomal protein of 170 kDa	1↑
Clasp1	CLIP-associating protein 1	1↑
Cracdl	Capping protein-inhibiting regulator of actin-like	3↑
Crmp1	Crmp1 protein (Dihydropyrimidinase-related protein 1)	2↑
Dnal1	Dynein axonemal light chain 1	1↓
Dync1li2	Cytoplasmic dynein 1 light intermediate chain 2 (Dynein light intermediate chain 2, cytosolic)	1↓
Ermn	Ermin (Juxtanodin) (JN)	1↓
Evl	Ena/VASP-like protein (Ena/vasodilator-stimulated phosphoprotein-like)	1↑
Gap43	Neuromodulin (Axonal membrane protein GAP-43) (Calmodulin-binding protein P-57) (Growth-associated protein 43)	1↑
Gas2l1	GAS2-like protein 1 (Growth arrest-specific protein 2-like 1)	1↑
Hook3	Protein Hook homolog 3 (mHK3)	1↓
Kif1b	Kinesin-like protein KIF1B	1↓
Limk1	LIM domain kinase 1 (Fragment)	2↑
Map1a	Microtubule-associated protein 1A (MAP-1A) [Cleaved into: MAP1A heavy chain; MAP1 light chain LC2]	1↑4↓
Map1b	Microtubule-associated protein 1B (MAP-1B) (MAP1(X)) (MAP1.2) [Cleaved into: MAP1B heavy chain; MAP1 light chain LC1]	3↑3↓
Map2	Microtubule-associated protein 2 (MAP-2)	8↑
Map4	Microtubule-associated protein 4 (MAP-4)	1↑1↓
Map6	Microtubule-associated protein 6 (MAP-6) (Stable tubule-only polypeptide) (STOP)	1↑
Mtmr1	Myotubularin-related protein 1 (Phosphatidylinositol-3,5-bisphosphate 3-phosphatase) (EC 3.1.3.95) (Phosphatidylinositol-3-phosphate phosphatase) (EC 3.1.3.64)	1↑
Nefh	Neurofilament heavy polypeptide (NF-H) (200 kDa neurofilament protein) (Neurofilament triplet H protein)	6↓
Nefm	Neurofilament medium polypeptide (NF-M) (160 kDa neurofilament protein) (Neurofilament 3) (Neurofilament triplet M protein)	2↓
Rufy3	Protein RUFY3	2↑
Sptbn1	Spectrin beta chain, non-erythrocytic 1 (Beta-II spectrin) (Embryonic liver fodrin) (Fodrin beta chain)	3↓
Tppp	Tubulin polymerization-promoting protein (TPPP) (EC 3.6.5.-) (25 kDa brain-specific protein) (TPPP/p25) (p25-alpha)	2↑
Tsc2	Tuberin	1↑
Wipf2	WAS/WASL-interacting protein family member 2 (WASP-interacting protein-related protein) (WIP-related protein)	1↑

One-hundred and thirteen differentially regulated phosphoproteins were integral membrane proteins, linked to membrane metabolism including ion channels, synapsis, vesicles, endocytosis, and exocytosis. Eighty seven were up-regulated, 18 down-regulated, and eight with both up- and down-regulated phosphorylation sites in P301S compared with WT mice (Table 4).

Table 4

List of altered phosphoproteins in P301S in comparison with WT mice related to membrane and membrane-linked proteins. The number of deregulated phosphosites and direction (hyper- or hypophosphorylation) are indicated for each protein.
Protein name		Protein location	Phosphosite deregulation
Adcy2	Adenylate cyclase type 2 (EC 4.6.1.1)	Membrane signaling	1↑
Adcy9	Adenylate cyclase type 9	Membrane signaling	1↑
Add1	Alpha-adducin (Erythrocyte adducin subunit alpha)	Membrane/cytoskeleton	1↑ 3↓
Afdn	Afadin (Afadin adherens junction formation factor) (Protein Af-6)	Cell junction/membrane	1↑
Als2	Alsin (Amyotrophic lateral sclerosis 2 protein homolog)	Vesicles Rab	3↑
Amph	Amphiphysin	Membrane/synapsis	2↑
Ank2	Ankyrin-2	Integral membrane protein	5↑
Ank3	Ankyrin-3 (Fragment)	Integral membrane protein	2↑1↓
Ankrd34a	Ankyrin repeat domain 34A	Integral membrane protein	2↑
Ankrd34b	Ankyrin repeat domain-containing protein 34B (Dendritic cell progenitor protein of 58 kDa)	Integral membrane protein	1↑
Apba1	Amyloid-beta A4 precursor protein-binding family A member 1 (Adapter protein X11alpha)	Vesicle trafficking	2↑
Arfgap1	ADP-ribosylation factor GTPase-activating protein 1	Golgi complex/vesicles	1↓
Arhgap23	Rho GTPase-activating protein 23 (Rho-type GTPase-activating protein 23)	Membrane signaling	1↓
Arhgef7	Rho guanine nucleotide exchange factor 7 (Beta-Pix)	Membrane signaling	1↓
Asap1	Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1	Membrane signaling	1↑
Basp1	Brain acid soluble protein 1 (22 kDa neuronal tissue-enriched acidic protein)	Membrane	2↓
Bsn	Protein bassoon	Synapsis	1↓4↑
Cacna1e	Voltage-dependent R-type calcium channel subunit alpha-1E (Brain calcium channel II) (BII)	Ion cannel/AMPA receptor	2↑
Cacng8	Voltage-dependent calcium channel gamma-8 subunit	Ion channel/AMPA receptor	1↑
Cadps	Calcium-dependent secretion activator 1	Secretion vesicles	1↓
Cadps2	Calcium-dependent secretion activator 2	Secretion vesicles	2↑
Cavin2	Caveolae-associated protein 2 (Cavin-2)	Membrane/vesicles	1↑
Clvs2	Clavesin-2 (Retinaldehyde-binding protein 1-like 2)	Vesicles/endosomes	1↑
Ctnna1	Catenin alpha-1 (102 kDa cadherin-associated protein) (Alpha E-catenin) (CAP102)	Membrane/cytoskeleton	1↑
Ctnna2	Catenin alpha-2 (Alpha N-catenin)	Membrane/vesicles	1↑
Cttn	Cttn protein (Src substrate cortactin)	Cytoskeleton/membrane/vesicles	1↑
Dennd1a	DENN domain-containing protein 1A (Fragment)	Membrane/vesicles	1↑
Dlg2	Disks large homolog 2 (Postsynaptic density protein PSD-93)	Synapsis	1↓
Dlg4	Disks large homolog 4	Synapsis	2↑
Dlgap4	Disks large-associated protein 4 (Dlgap4 protein)	Synapsis	1↑
Dmd	Dystrophin (Dystrophin Dp71a)	Membranes	1↑
Dmtn	Dematin (Dematin actin-binding protein)	Membrane/cytoskeleton	1↓
Dmxl2	DmX-like protein 2 (Rabconnectin-3)	Synapsis	3↓
Dnal1	Dynein axonemal light chain 1	Cytoskeleton/membranes	1↓
Dnm1l	Dynamin-1-like protein (EC 3.6.5.5)	Membrane/vesicles	1↑
Dnm3	Dynamin-3 (EC 3.6.5.5)	Membrane/vesicles	2↑
Dpysl2	Dihydropyrimidinase-related protein 2 (DRP-2)	Membrane signaling	4↑
Dpysl3	Dihydropyrimidinase-related protein 3 (DRP-3)	Membrane/cytoskeleton	2↑
Dpysl5	Dihydropyrimidinase-related protein 5 (DRP-5)	Membrane/cytoskeleton	8↑
Eef1akmt1	EEF1A lysine methyltransferase 1 (EC 2.1.1.-) (N(6)-adenine-specific DNA methyltransferase 2) (Protein-lysine N-methyltransferase N6amt2)	Vesicles	1↓
Ehd1	EH domain-containing protein 1 (PAST homolog 1) (mPAST1)	Membrane/endosomes	1↓
Ehd3	EH domain-containing protein 3	Membrane/endosomes	1↓
Epb41l3	Band 4.1-like protein 3 (4.1B) (Differentially expressed in adenocarcinoma of the lung protein 1) (DAL-1) (DAL1P) (mDAL-1)	Synapses	3↑1↓
Epn1	Epsin-1 (EPS-15-interacting protein 1) (Intersectin-EH-binding protein 1) (Ibp1)	Membranes/endocytosis	1↑1↓
Fam126b	Protein FAM126B	Membrane signaling/vesicles	1↑
Fam160a2	FTS and Hook-interacting protein	Membrane signalling/vesicles	1↑
Farp1	FERM, ARHGEF and pleckstrin domain-containing protein 1 (FERM, RhoGEF and pleckstrin domain-containing protein 1)	Membrane signalling/synapsis	2↑
Frmd4a	FERM domain-containing protein 4A	Membrane/cytoskeleton	2↑
Gabra1	Gamma-aminobutyric acid receptor subunit alpha-1 (GABA(A) receptor subunit alpha-1)	Membrane/GABA receptor	1↑
Gabrg2	Gamma-aminobutyric acid receptor subunit gamma-2 (GABA(A) receptor subunit gamma-2)	Membrane/GABA receptor	1↑
Gja1	Gap junction alpha-1 protein (Connexin-43) (Cx43) (Gap junction 43 kDa heart protein)	Gap junction	2↑
Grm5	Metabotropic glutamate receptor 5 (mGluR5)	Membrane/GLU receptor	2↑
Hcn2	Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2 (Brain cyclic nucleotide-gated channel 2) (BCNG-2)	Ion channel/membrane	1↑
Hepacam	Hepatocyte cell adhesion molecule (Protein hepaCAM)	Membrane	1↑
Iqsec1	IQ motif and SEC7 domain-containing protein 1	Vesicles	2↑
Iqsec2	ARF6 guanine nucleotide exchange factor IQArfGEF (IQ motif and SEC7 domain-containing protein 2)	Synapsis	3↑
Itsn1	Intersectin-1 (EH and SH3 domains protein 1)	Membrane/vesicle/synapsis	1↑
Kcnb1	Potassium voltage-gated channel subfamily B member 1 (Voltage-gated potassium channel subunit Kv2.1) (mShab)	Ion channel/membrane	1↑
Kcnq2	Potassium voltage-gated channel subfamily KQT member 2 (Fragment)	Ion channel/membrane	1↑
Kctd12	BTB/POZ domain-containing protein KCTD12	Membrane/GABA receptor	1↑
Kif1a	Kinesin-like protein KIF1A (Kinesin-related microtuble-based motor protein)	Axonal transport/vesicles	1↑
Kif2a	Kinesin-like protein	Axonal transport/vesicles	1↑
Klc1	Kinesin light chain	Axonal transport/vesicles	4↑
Klc2	Kinesin light chain	Axonal transport/vesicles	2↑
Klc4	Kinesin light chain 4 (KLC 4) (Kinesin-like protein 8)	Axonal transport/vesicles	1↑
Lrrc7	Leucine-rich repeat-containing protein 7 (Densin-180) (Densin) (Protein LAP1)	Synapsis	1↑
Nsf	Vesicle-fusing ATPase (EC 3.6.4.6)	Vesicles	1↓
Ociad1	OCIA domain-containing protein 1	Vesicles/endosomes	1↑
Pcdh1	Protocadherin 1 (Fragment)	Membrane protein	2↑
Pclo	Protein piccolo (Aczonin) (Brain-derived HLMN protein) (Multidomain presynaptic cytomatrix protein)	Synapsis	4↑2↓
Pip4p1	Type 1 phosphatidylinositol 4,5-bisphosphate 4-phosphatase (Type 1 PtdIns-4,5-P2 4-Ptase) (EC 3.1.3.78)	Membrane/cholesterol	1↑
Pitpnm1	Membrane-associated phosphatidylinositol transfer protein 1 (Drosophila retinal degeneration B homolog 1) (RdgB1) (Mpt-1)	Membrane	4↑
Prrt3	Proline-rich transmembrane protein 3	Membrane	2↑
Rab11fip5	Rab11 family-interacting protein 5	Traffick vesicles	3↑
Rab3gap2	Rab3 GTPase-activating protein non-catalytic subunit	Vesicle transport	1↑
Rabep1	Rab GTPase-binding effector protein 1 (Rabaptin-5) (Rabaptin-5alpha)	Vesicles	2↓
Ralbp1	RalA-binding protein 1	Endocytosis	↓
Ralgapb	Ral GTPase-activating protein subunit beta (Fragment)	Vesicles	1↑
Ranbp3	Ran-binding protein 3 (RanBP3)	Transport vesicles	1↓
Rasgrf2	Ras-specific guanine nucleotide-releasing factor 2 (Ras-GRF2) (Ras guanine nucleotide exchange factor 2)	Synapses	1↑
Rbsn	Rabenosyn-5 (FYVE finger-containing Rab5 effector protein rabenosyn-5) (RAB effector RBSN)	Endosomes/vesicles	1↓
Relch	RAB11-binding protein RELCH	Endosomes/cytoskeleton/Golgi	3↑2↓
Rtn3	Reticulon-3	Secretory vesicles	4↓
Rtn4	Reticulon-4 (Neurite outgrowth inhibitor) (Nogo protein)	Membrane trafficking/secretory vesicles	3↑
Scn2a	Sodium channel protein	Ion channel/membrane	2↑
Septin8	Septin-8	Synaptic vesicles	1↑
Sgip1	SH3-containing GRB2-like protein 3-interacting protein 1 (Endophilin-3-interacting protein)	Vesicles/endocytosis	1↑
Shisa6	Protein shisa-6 (Fragment)	Membrane/receptor AMPA	2↑
Slc12a5	Solute carrier family 12 member 5 (Electroneutral potassium-chloride cotransporter 2) (K-Cl cotransporter 2)	Membrane transporter	1↑
Slc20a2	Sodium-dependent phosphate transporter 2 (Phosphate transporter 2) (PiT-2) (Solute carrier family 20 member 2)	Membrane transporter	2↑
Slc6a17	Sodium-Dependent Neutral Amino Acid Transporter SLC6A17	Synapsis	1↑
Slc7a2	Cationic amino acid transporter 2 (CAT-2) (CAT2) (20.5)	Membrane transporter	1↑
Slc7a8	Large neutral amino acids transporter small subunit 2 (L-type amino acid transporter 2) (mLAT2) (Solute carrier family 7 member 8)	Membrane transporter	1↑
Smap2	Stromal membrane-associated protein 2 (Stromal membrane-associated protein 1-like)	Endocytosis	1↑
Snap25	Synaptosomal-associated protein 25 (SNAP-25) (Super protein) (SUP) (Synaptosomal-associated 25 kDa protein)	Synapsis	1↑
Snap91	Clathrin coat assembly protein AP180 (91 kDa synaptosomal-associated protein)	Synapsis	1↑
Srcin1	SRC kinase-signaling inhibitor 1	Synapsis/cell signalling	3↑
Svop	Synaptic vesicle 2-related protein (SV2-related protein)	Synapsis	1↑
Syn1	Synapsin-1 (Synapsin I)	Synapsis	5↑
Syn3	Synapsin-3 (Synapsin III)	Synapsis	3↑
Synj1	Phosphoinositide 5-phosphatase (EC 3.1.3.36)	synapsis	1↑
Tanc2	Protein TANC2 (Tetratricopeptide repeat, ankyrin repeat and coiled-coil domain-containing protein 2)	Synapsis	2↑
Tbc1d10b	TBC1 domain family member 10B (Protein wz3-85)	Vesicle transport	1↑
Tbc1d22b	TBC1 domain family, member 22B	Vesicle transport	1↑
Tjp1	Tight junction protein ZO-1 (Tjp1 protein)	Membrane	1↓
Tjp2	Tight junction protein ZO-2	Membrane	1↑
Tmcc1	Transmembrane and coiled-coil domains protein 1	Membrane	1↑
Tmcc2	Transmembrane and coiled-coil domains protein 2	Membrane	1↑
Tns1	Tensin 1 (Fragment)	Membrane	2↑
Tom1	Target of Myb protein 1	Vesicles	2↑
Tprg1l	Tumor protein p63-regulated gene 1-like protein	Synapsis	1↑
Unc13a	Protein unc-13 homolog A (Munc13-1)	Vesicles/synapsis	1↓1↑
Vamp2	Vesicle-associated membrane protein 2 (VAMP-2) (Synaptobrevin-2)	Synapsis	1↑
Vdac1	Voltage-dependent anion-selective channel protein 1 (VDAC-1) (mVDAC1) (Outer mitochondrial membrane protein porin 1)	Ion channel/mitochondrial membrane protein	1↓

Forty dysregulated phosphoproteins were linked to DNA and RNA metabolism, RNA splicing, and protein synthesis; 37 phosphoproteins were down-regulated, and 3 up-regulated in P301S compared with WT mice (Table 5).

Table 5

List of altered phosphoproteins in P301S in comparison with WT mice related to DNA, RNA metabolism, and protein synthesis. The number of dysregulated phosphosites and direction (hyper- or hypophosphorylation) are indicated for each protein.
Protein name		Protein function	Phosphosite deregulation
Aarsd1	Alanyl-tRNA editing protein Aarsd1 (Alanyl-tRNA deacylase alaX) (AlaX) (AlaXp-II) (Alanyl-tRNA synthetase domain-containing protein 1)	DNA/RNA	1↓
Ccar2	Cell cycle and apoptosis regulator protein 2 (Cell division cycle and apoptosis regulator protein 2)	DNA	1↓
Cic	Protein capicua homolog	DNA/RNA	1↓
Cwc15	Spliceosome-associated protein CWC15 homolog (Embryonic development factor 1) (mED1)	DNA/RNA	2↓
Dhx32	Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX32 (Fragment)	DNA/RNA	1↓
Eif2s2	Eukaryotic translation initiation factor 2 subunit 2	RNA/protein synthesis	1↓
Eif3j1	Eukaryotic translation initiation factor 3 subunit J-A (eIF3j-A) (Eukaryotic translation initiation factor 3 subunit 1-A) (eIF-3-alpha-A) (eIF3 p35)	RNA/protein synthesis	2↓
Eif5b	Eukaryotic translation initiation factor 5B (eIF-5B) (EC 3.6.5.3) (Translation initiation factor IF-2)	RNA/protein synthesis	3↓
Hdac2	Histone deacetylase 2 (HD2) (EC 3.5.1.98)	DNA	2↓
Hdgf	Hepatoma-derived growth factor (HDGF)	DNA	1↓
Hdgfl3	Hepatoma-derived growth factor-related protein 3 (HRP-3)	DNA/RNA	2↓
Hnrnpu	Heterogeneous nuclear ribonucleoprotein U (hnRNP U) (Scaffold-attachment factor A) (SAF-A)	DNA/RNA	1↓
Kdm1a	Lysine-specific histone demethylase 1A (Fragment)	DNA	1↓
Nab2	NGFI-A-binding protein 2	RNA transcription	2↓
Ncl	Nucleolin (Protein C23)	RNA	4↓
Ncoa5	Nuclear receptor coactivator 5 (NCoA-5) (Coactivator independent of AF-2) (CIA)	Estrogen receptor nucleus	1↓
Nsrp1	Nuclear speckle splicing regulatory protein 1 (Coiled-coil domain-containing protein 55) (Nuclear speckle-related protein 70) (NSrp70)	DNA/RNA	2↓
Pcbp1	Poly(rC)-binding protein 1 (Alpha-CP1) (Heterogeneous nuclear ribonucleoprotein E1) (hnRNP E1)	RNA	1↑
Pnn	Pinin	DNA/RNA	1↓
Psd3	PH and SEC7 domain-containing protein 3	DNA/RNA	1↓
Ptges3	Prostaglandin E synthase 3 (EC 5.3.99.3)	Chaperone/DNA metabolism	1↓
Ralgapa1	Ral GTPase-activating protein subunit alpha-1	RNA transcription	1↑
Rbm10	RNA-binding protein 10 (RNA-binding motif protein 10)	RNA	1↓
Rbm25	RNA-binding protein 25 (RNA-binding motif protein 25)	RNA	1↓
Rbm33	RNA-binding protein 33	RNA	1↓
Rplp1	60S acidic ribosomal protein P1	RNA protein synthesis	1↓
Rplp2	60S acidic ribosomal protein P2	RNA protein synthesis	2↓
Safb	Scaffold attachment factor B1 (SAF-B1)	DNA	1↓
Sltm	SAFB-like transcription modulator (Modulator of estrogen-induced transcription)	Protein synthesis	1↑
Smarcc2	SWI/SNF complex subunit SMARCC2	DNA	1↓
Smn1	Survival motor neuron protein	DNA	1↓
Srsf1	Serine/arginine-rich splicing factor 1 (Splicing factor, arginine/serine-rich 1)	RNA splicing	2↓
Srsf10	Serine/arginine-rich-splicing factor 10	RNA splicing	2↓
Srsf6	Serine/arginine-rich splicing factor 6 (Pre-mRNA-splicing factor SRP55) (Splicing factor, arginine/serine-rich 6)	RNA splicing	1↓
Srsf7	Serine/arginine-rich splicing factor 7 (Splicing factor, arginine/serine-rich 7)	RNA splicing	1↓
Supt6h	Transcription elongation factor SPT6	Protein synthesis	1↓
Tceal3	Transcription elongation factor A protein-like 3 (TCEA-like protein 3)	Protein synthesis	1↓
Thrap3	Thyroid hormone receptor-associated protein 3	DNA	1↓
Top2b	DNA topoisomerase 2-beta (EC 5.6.2.2) (DNA topoisomerase II, beta isozyme)	DNA	1↓
Tra2b	Transformer-2 protein homolog beta (TRA-2 beta) (TRA2-beta)	RNA splicing	1↓

Sixteen differentially regulated phosphoproteins were components of the cellular degrading systems, autophagy, and UPS. Five phosphoproteins were up-regulated and 11 down-regulated (Table 6).

Table 6

List of altered phosphoproteins in P301S in comparison with WT mice related to autophagy and the ubiquin-proteasome system (UPS). The number of dysregulated phosphosites and direction (hyper- or hypophosphorylation) are indicated for each protein.
Protein name		Protein function	Phosphosite deregulation
Atg2b	Autophagy-related protein 2 homolog B	Autophagy	1↑
Atg9a	Autophagy-related protein 9	Autophagy	1↑
Cand1	Cullin-associated NEDD8-dissociated protein 1 (Cullin-associated and neddylation-dissociated protein 1) (p120 CAND1)	UPS	2↓
Dcaf11	DDB1- and CUL4-associated factor 11	UPS	2↓
Fbxo41	F-box only protein 41	UPS	1↓
Hectd4	HECT domain E3 ubiquitin protein ligase 4	UPS	1↓
Lmbrd1	Lysosomal cobalamin transport escort protein LMBD1 (LMBD1) (LMBR1 domain-containing protein 1) (Protein N90b)	Lysosomes	1↓
Lrsam1	E3 ubiquitin-protein ligase LRSAM1 (EC 2.3.2.27)	UPS	1↓
Mgrn1	E3 ubiquitin-protein ligase MGRN1 (EC 2.3.2.27)	UPS	1↓
Nedd4l	HECT-type E3 ubiquitin transferase (EC 2.3.2.26)	UPS	2↑
Psmd11	26S proteasome non-ATPase regulatory subunit 11 (26S proteasome regulatory subunit RPN6) (26S proteasome regulatory subunit S9) (26S proteasome regulatory subunit p44.5)	UPS	1↑
Stub1	E3 ubiquitin-protein ligase CHIP (EC 2.3.2.27)	UPS	1↓
Trim2	Tripartite motif-containing protein 2 (EC 2.3.2.27)	UPS	1↓
Trim9	E3 ubiquitin-protein ligase TRIM9	UPS	2↑
Uba1	Ubiquitin-like modifier-activating enzyme 1 (EC 6.2.1.45)	UPS	1↓
Ube2o	(E3-independent) E2 ubiquitin-conjugating enzyme UBE2O (EC 2.3.2.24)	UPS	1↓
Usp5	Ubiquitin carboxyl-terminal hydrolase (EC 3.4.19.12)	UPS	3↓
Usp9x	Ubiquitinyl hydrolase 1 (EC 3.4.19.12)	UPS	1↓

Nine dysregulated phosphoproteins were phosphatases, five were up-regulated, and four down-regulated in P301S when compared with WT mice (Table 7).

Table 7

List of altered phosphoproteins in P301S in comparison with WT mice related to phosphatases and their activity. The number of dysregulated phosphosites and direction (hyper- or hypophosphorylation) are indicated for each protein.
Protein name		Protein function	Phosphosite deregulation
Elfn2	Protein phosphatase 1 regulatory subunit 29 (Extracellular leucine-rich repeat and fibronectin type III domain-containing protein 2) (Leucine-rich repeat and fibronectin type-III domain-containing protein 6) (Leucine-rich repeat-containing protein 62)	Cytoskeleton	1↑
Pip4p1	Type 1 phosphatidylinositol 4,5-bisphosphate 4-phosphatase (Type 1 PtdIns-4,5-P2 4-Ptase) (EC 3.1.3.78)	Membrane/cholesterol	1↑
Plpp6	Phospholipid phosphatase 6 (EC 3.1.3.-)	Lipid metabolism	1↑
Ppme1	Protein phosphatase methylesterase 1 (PME-1) (EC 3.1.1.89)	Demetylates proteins (phosphatases)	1↓
Ppp1r1a	Protein phosphatase 1 regulatory subunit 1A (Protein phosphatase inhibitor 1) (I-1) (IPP-1)	Phosphatase	1↑
Ppp1r9a	Protein phosphatase 1, regulatory subunit 9A (Fragment)	Glycogen metabolism/phosphatase	1↓
Ppp2r5e	Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit	Phosphatase	2↑
Ppp3cb	Serine/threonine-protein phosphatase (EC 3.1.3.16)	Phosphatase	1↓
Ptprz1	Receptor-type tyrosine-protein phosphatase zeta (R-PTP-zeta) (EC 3.1.3.48)	Oligodendrocytes	1↓

Thirty-nine differentially regulated phosphoproteins (22 up-regulated, 17 down-regulated, and one with up- and down-regulated phosphorylation sites) were involved in cell signalling, energy metabolism, lipid metabolism, and carbohydrate metabolism, among other functions (Table 8). The classification is considered instrumental as many proteins are involved in different pathways.

Table 8

List of altered phosphoproteins in P301S in comparison with WT mice related to energy metabolism, cell signalling, Golgi apparatus, carbohydrates, and lipids. The number of dysregulated phosphosites and direction (hyper- or hypophosphorylation) are indicated for each protein.
Protein names		Protein function	Phosphosite deregulation
Acaca	Acetyl-CoA carboxylase 1 (ACC1) (EC 6.4.1.2)	Fatty acid synthesis	1↓
Agfg1	Arf-GAP domain and FG repeat-containing protein 1	Nucleoporin	2↑
Akr7a2	Aflatoxin B1 aldehyde reductase member 2 (EC 1.1.1.n11)	Energy metabolism	1↑
Amer2	APC membrane recruitment protein 2 (Amer2) (Protein FAM123A)	Negative regulator WNT	1↑
Ampd2	AMP deaminase (EC 3.5.4.6)	Purine metabolism	1↑
Atl1	Atlastin-1 (EC 3.6.5.-)	Golgi	1↑
Bclaf1	Bcl-2-associated transcription factor 1 (Btf)	Cell death	1↓
C2cd2l	Phospholipid transfer protein C2CD2L (C2 domain-containing protein 2-like) (Transmembrane protein 24)	Lipid transport	1↑
Cnksr2	Connector enhancer of kinase suppressor of ras 2 (Connector enhancer of KSR 2) (CNK homolog protein 2) (CNK2)	Cell signalling	1↑
Coq9	Ubiquinone biosynthesis protein COQ9, mitochondrial	Energy metabolism	1↓
Dagla	Diacylglycerol lipase-alpha (DAGL-alpha) (DGL-alpha) (EC 3.1.1.-)	DAG, lipids	1↑
Dock7	Dedicator of cytokinesis protein 7	Cell signalling	3↑
Fgf12	Fibroblast growth factor 12 (FGF-12) (Fibroblast growth factor homologous factor 1) (FHF-1) (Myocyte-activating factor)	FGF	1↓
Hsph1	Heat shock protein 105 kDa (42 degrees C-HSP) (Heat shock 110 kDa protein) (Heat shock-related 100 kDa protein E7I) (HSP-E7I)	Chaperone	1↓
Ipo7	Importin-7 (Imp7) (Ran-binding protein 7) (RanBP7)	Nuclear transport	2↓
Ldhb	L-lactate dehydrogenase B chain (LDH-B) (EC 1.1.1.27)	Energy metabolism	1↓
Lgalsl	Galectin-related protein (Galectin-related protein A) (Lectin galactoside-binding-like protein A)	Carbohydrate metabolism	5↓
Mbp	Myelin basic protein	Myelin	2↓
Mff	Mitochondrial fission factor	Energy metabolism	1↓
Mief1	Mitochondrial dynamics protein MID51	Energy metabolism	1↓
Mtx3	Metaxin	Mitochondria/transport	1↑
Myo18a	Unconventional myosin-XVIIIa (Molecule associated with JAK3 N-terminus) (MAJN) (Myosin containing a PDZ domain) (Surfactant protein receptor SP-R210) (SP-R210)	Golgi/trafficking	1↓
Pacs1	Phosphofurin acidic cluster sorting protein 1 (PACS-1)	Trans-Golgi	1↑
Pdap1	28 kDa heat- and acid-stable phosphoprotein (PDGF-associated protein) (PAP) (PDGFA-associated protein 1) (PAP1)	Cell signalling	1↑1↓
Pde4a	cAMP-specific 3',5'-cyclic phosphodiesterase 4A (EC 3.1.4.53)	Cell signaling	1↓
Pex5l	PEX5-related protein	Peroxisome	1↑
Pgam1	Phosphoglycerate mutase 1 (EC 5.4.2.11) (EC 5.4.2.4)	Glycogen metabolism	1↓
Pikfyve	1-phosphatidylinositol 3-phosphate 5-kinase (Phosphatidylinositol 3-phosphate 5-kinase) (EC 2.7.1.150)	Golgi/transport	1↑
Plekha5	Pleckstrin homology domain-containing, family A member 5	Lipid metabolism	1↑
Plpp6	Phospholipid phosphatase 6 (EC 3.1.3.-)	Lipid metabolism	1↑
Ptprz1	Receptor-type tyrosine-protein phosphatase zeta (R-PTP-zeta) (EC 3.1.3.48)	Oligodendrocytes	1↓
Rap1gap	Rap1 GTPase-activating protein 1 (Rap1GAP) (Rap1GAP1) (ARPP-90)	Cell signalling	↓↑
Rapgef2	Cyclic nucleotide ras GEF (Neural RAP guanine nucleotide exchange protein) (PDZ domain-containing guanine nucleotide exchange factor 1) (RA-GEF-1) (Rap guanine nucleotide exchange factor 2) (Ras/Rap1-associating GEF-1)	Cell signalling	1↑
Rb1cc1	RB1-inducible coiled-coil protein 1 (Coiled-coil-forming protein 1) (FAK family kinase-interacting protein of 200 kDa) (FIP200) (LaXp180)	Cell signalling	2↑
Rgs14	Regulator of G-protein signaling 14 (RGS14) (RAP1/RAP2-interacting protein) (RPIP1)	Cell signalling	1↑
Sec31a	Protein transport protein Sec31A (SEC31-like protein 1) (SEC31-related protein A)	Endoplasmic reticulum/transportr	1↓
Septin5	Septin	Transport	1↑
Spred1	Sprouty-related, EVH1 domain-containing protein 1 (Spred-1)	Cell signalling	1↑
Tnik	TRAF2 and NCK interacting kinase (Traf2 and NCK-interacting protein kinase)	Cell signalling	1↓
Tom1l2	TOM1-like protein 2 (Target of Myb-like protein 2)	Transport	2↑

Phosphoprotein Interactomes In P301s Mice

Network analysis was performed submitting the corresponding protein IDs to the STRING. A schematic representation is shown in Fig. 6. Abbreviations and quantitative values are detailed in Supplementary Table 1. The image illustrates the complementary phosphoprotein network dysregulated in P301S tauopathy. There is a close interaction between proteins of the membranes and cytoskeleton, and proteins with kinase activity.

P301S transgenic mice develop hyper-phosphorylated tau deposits in neurons with the appearance of pre-tangles in the cerebral cortex, amygdala, hippocampus, striatum, and thalamus; and neuropil threads in the cerebral cortex, and plexiform layers of the hippocampus. Tau deposits are positive with phospho-specific anti-tau antibodies AT8 (Ser202/Thr205), PHF1 (Ser396/404), Tau-P Ser422, Tau 100 (Tau-P Thr212/Ser214), and antibodies against epitopes within amino acids 312–322 (conformational antibody MC1). Truncated tau at the aspartic acid 421 (Tau C-3) is also expressed in a subset of neurons at advanced stages of the tauopathy [40]. Affected neurons in P301S transgenic mice also contain nitrated tau (Tau-N Tyr29). Although there is a positive 4Rtau background, neuronal tau deposits do not show 4Rtau immunoreactive enhancement. Tau phosphorylation is accompanied by activation of p38, SAPK/JNK, and SRC kinases, as identified with the antibodies p38-P Thr180/182, SAPK/JNK-P Thr183/Thr185, and SRC-P Tyr416, respectively, but GSK-3β-P Ser9 immunoreactivity is negative. Affected neurons also show rare granular casein kinase δ immunoreactivity. In addition to phospho-tau, neuronal deposits are strongly immunoreactive with the MAP2-P Thr1620/1623 antibody. Markers of endoplasmic reticulum stress and autophagy (eIF2α-P Ser51, IRE1-P Ser 274, LC3, and LAMP) are negative. The battery of antibodies used to characterize P301S mice, mainly oriented to learning about abnormal phosphorylation of specific substrates, and activation of related kinases, was based on the previous systematic approach applied to the study of the characteristics of tau seeding and spreading following intrahippocampal inoculation of sarkosyl-insoluble fractions enriched in tau from human AD and other tauopathies [30, 41].

Based on these results, label-free (phospho)proteomics and SWATH-MS was used to identify differential protein phosphorylation between P301S and WT mice.

Previous phosphoproteomics analysis in AD transgenic models was mainly focused on transgenic mice generating β-amyloid deposits; (phospho)protemics examination in tau transgenic mice is more limited. Phosphoproteomics in double-APPswe/PS1ΔE9 mouse (APP/PS1), double-mutant KM670/671NL/V717F of human APP (TgCRND8), and APPSwDI mice lacking inducible nitric oxide synthase (CVN-AD) mice, all of them resulting in β-amyloid deposition, have shown altered regulation of protein phosphorylation mainly involving synaptic proteins; other targets include gap junctions, activation of astrocytes, mitochondria, vesicle trafficking, innate immune pathways, connexin 43, CREB signalling, and cytoskeletal dynamics [24–27, 29]. Phosphoproteomics was carried out in parallel in five transgenic mouse models of AD, including PSEN1dE9, PS2, double-mutant APP770 (KM670/671NL), 5xFAD transgenic mice expressing the mutant human Swedish KM670/671NL, Florida (I716V) and London (V717I) triple mutations, and PS1 harbouring double mutations (M146L and L285V) [26]. PKC, mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5) were identified as the first group of kinases that regulate phosphorylation state of the core network proteins; casein kinase 2 (CSKII), receptor interacting serine/threonine-protein kinase 1/3 (RIPK1/3), cyclin-dependent kinase 5/6 (CDK 5/6), and protein kinase C-like 1 (PKN1) made up the second group. The third group was comprised of Ca²⁺/calmodulin-dependent kinase (CaMKI/II), protein kinase D (PRKD), Lck/Yes-related novel protein tyrosine kinase (LYN), and other kinases linked to only one protein [26]. Systems biology suggested that over-activated kinases protein kinases C (PKC) and calmodulin-dependent kinases (CAMK2) initiate synapse pathology through phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) [26]. Altered age-dependent immune responses and synaptic dysfunction have also been reported following proteomic analysis and bioinformatics analysis in ADLP^APT mice which carry three human transgenes, including β-amyloid precursor protein, presenilin-1 and tau, with six mutations [28].

Early synaptic pathology, changes in the synaptic proteome, altered neuroinflammatory gene regulation, oxidative stress, mitochondrial dysfunction, and brain lipid modifications are characteristic alterations in P301S transgenic mice [40, 42–45]. Increased CaMKII phosphorylation, and S-nitrosylation of E3 ubiquitin-protein ligase RNF213 (RNF-213), have also been identified in P301S mice [46]. Previous phosphoprotemics in P301S mice disclosed dysregulated phosphorylation of adducin 2 (ADDB), neurofilament heavy polypeptide (NFH), neurofilament light polypeptide (NFL), spectrin alpha non-erythrocytic (SPTA2), brain abundant membrane attached signal protein 1 (BASP1), clathrin (CLH), myristotelated alanine-rich C-kinase substrate (MARCS), neuromodulin (NEUM), serine/arginine repetitive matrix 2 (SRRM2), and MARCS- like 1 (Marcsl1) [26].

The present findings reveal wide dysregulated protein phosphorylation in P301S transgenic mice that involves numerous proteins associated with membranes and cell membrane trafficking, synapses, neurotransmitter receptors, membrane vesicles linked to endo- and exocytosis, cytoplasmic vesicles, and the cytoskeleton, together with altered phosphorylation of a large number of kinases targeting various substrates. The targets are similar to those identified in sAD and GGT [21, 23], thus suggesting that abnormal tau, as exemplified by mutant P301S tau in transgenic mice, is associated with dysregulated protein phosphorylation of key components of membranes and the cytoskeleton, and mediated by abnormal activation of kinases. Alterations of these particular proteins implicate abnormal membrane signalling and cytoskeletal function, altered neurotransmission, and membrane and cytoplasmic vesicle metabolism.

In addition, proteins modulating DNA and RNA metabolism, RNA splicing, and protein syntyhesis largely appear to be hypo-phosphorylated. Modifications of these groups of proteins may result in altered DNA, RNA, and protein metabolism functions. Other pathways modulating energy metabolism, cell signalling, Golgi apparatus, carbohydrates, and lipids are also targets of dysregulated protein phosphorylation in P301S mice.

Further studies are needed to learn about the molecular signals connecting abnormal tau load with the astonishing dysregulated protein phosphorylation in tauopathies.

Financial disclosure and conflict of interests

No relevant data.

Ethics approval and consent to participate

Not applicable

Availability of data and materials:

All data generated or analyzed during this study are included in this published article and its supplementary information files; MS data and search results files were deposited in the Proteome Xchange Consortium via the JPOST partner repository (https://repository.jpostdb.org, accessed on 1 March 2022) (Okuda, S.; Watanabe, Y. et al. jPOSTrepo: An international standard data repository for proteomes. Nucleic Acids Res. 2017) with the identifier PXD031961 for ProteomeXchange and JPST001509 for jPOST. (For reviewers:

https://repository.jpostdb.org/preview/1738288420621e76ca4fcef; Access key 8489)

Funding

The project leading to these results received funding from the “la Caixa” Foundation (ID 100010434) under the agreement LCF/PR/HR19/52160007 to IF. We thank CERCA Programme/Generalitat de Catalunya for institutional support. The Proteomics Platform of Navarrabiomed is a member of Proteored (PRB3-ISCIII), and is supported by grant PT17/0019/009 to JFI, of the PE I+D+I 2013-2016 funded by ISCIII and FEDER. Part of this work was funded by a grant from the Spanish Ministry of Science Innovation and Universities (Ref. PID2019-110356RB-I00) to JFI and ES, and the Department of Economic and Business Development of the Government of Navarra (Ref. 0011-1411-2020-000028) to ES.

Author’s contributions

IF, PAB, and ES designed the study; PAB prepared the colonies; JFI, KA, and ES performed the proteomic analysis; IF, PAB, JAR, KA, PC, ML, JFI, and ES analyzed and interpreted the data; IF, PAB, and ES wrote the manuscript. All authors critically revised the manuscript.

Acknowledgements

We wish to thank Margarita Carmona for technical support and Benjamin Torrejón-Escribano (Advanced Light Microscopy Unit, Bellvitge Campus, Scientific and Technical Facility, University of Barcelona) for the confocal microscopy studies. We are grateful to T. Yohannan for editorial assistance.

Consent for publication

Not applicable

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Supplementarytable1.Differentialphosphoproteomics.xlsx
Supplementary Table 1: Differential phosphoproteomics detected in P301S transgenic mice.
Suppl.Table2.InteractionsinteractorsP301SvsWT.xlsx
Supplementary Table 2: List of interacting dysregulated phosphoproteins depicted in Figure 6 and their quantitative values.

Dysregulated protein phosphorylation in tauopathy

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods